@misc{hill_murphy_polkoff_edwards_walker_moatti_greenbaum_piedrahita_2024, title={A gene edited pig model for studying LGR5<SUP>+</SUP> stem cells: implications for future applications in tissue regeneration and biomedical research}, volume={6}, ISSN={["2673-3439"]}, DOI={10.3389/fgeed.2024.1401163}, abstractNote={Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5 + subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5 + cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5 + pig model in exploring the role of LGR5 + cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.}, journal={FRONTIERS IN GENOME EDITING}, author={Hill, Amanda B. T. and Murphy, Yanet M. and Polkoff, Kathryn M. and Edwards, Laura and Walker, Derek M. and Moatti, Adele and Greenbaum, Alon and Piedrahita, Jorge A.}, year={2024}, month={Jun} }
 @article{schaaf_polkoff_carter_stewart_sheahan_freund_ginzel_snyder_roper_piedrahita_et al._2023, title={A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease}, volume={37}, ISSN={["1530-6860"]}, DOI={10.1096/fj.202300223R}, abstractNote={Abstract}, number={6}, journal={FASEB JOURNAL}, author={Schaaf, Cecilia R. and Polkoff, Kathryn M. and Carter, Amber and Stewart, Amy S. and Sheahan, Breanna and Freund, John and Ginzel, Joshua and Snyder, Joshua C. and Roper, Jatin and Piedrahita, Jorge A. and et al.}, year={2023}, month={Jun} }
 @article{zhang_wang_lanzoni_wauthier_simpson_ezzell_allen_suitt_krolik_jhirad_et al._2023, title={A postnatal network of co-hepato/pancreatic stem/progenitors in the biliary trees of pigs and humans}, volume={8}, ISSN={["2057-3995"]}, DOI={10.1038/s41536-023-00303-5}, abstractNote={Abstract}, number={1}, journal={NPJ REGENERATIVE MEDICINE}, author={Zhang, Wencheng and Wang, Xicheng and Lanzoni, Giacomo and Wauthier, Eliane and Simpson, Sean and Ezzell, Jennifer Ashley and Allen, Amanda and Suitt, Carolyn and Krolik, Jonah and Jhirad, Alexander and et al.}, year={2023}, month={Aug} }
 @article{liu_paudel_flowers_piedrahita_wang_2023, title={Adrenomedullin Stimulates Proliferation, Migration and Adhesion of Porcine Trophectoderm Cells Via CALCRL-AKT-TSC2-MTORC1 Cell Signaling Pathway}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.040}, abstractNote={Abstract}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Liu, Bangmin and Paudel, Sudikshya and Flowers, William L. and Piedrahita, Jorge A. and Wang, Xiaoqiu}, year={2023}, month={May} }
 @article{gonzalez_mitchell-dick_blondel_fanous_hull_oh_moller-tank_rivera_piedrahita_asokan_2023, title={Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery}, volume={9}, ISSN={["1750-2799"]}, DOI={10.1038/s41596-023-00875-y}, journal={NATURE PROTOCOLS}, author={Gonzalez, Trevor J. and Mitchell-Dick, Aaron and Blondel, Leo O. and Fanous, Marco M. and Hull, Joshua A. and Oh, Daniel K. and Moller-Tank, Sven and Rivera, Ruth M. Castellanos and Piedrahita, Jorge A. and Asokan, Aravind}, year={2023}, month={Sep} }
 @article{liu_paudel_flowers_piedrahita_wang_2023, title={Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway}, volume={4}, ISSN={["1438-2199"]}, DOI={10.1007/s00726-023-03265-6}, abstractNote={Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10–7 M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.}, journal={AMINO ACIDS}, author={Liu, Bangmin and Paudel, Sudikshya and Flowers, William L. and Piedrahita, Jorge A. and Wang, Xiaoqiu}, year={2023}, month={Apr} }
 @article{howe_cone_piedrahita_spang_fisher_2022, title={Age- and Sex-Specific Joint Biomechanics in Response to Partial and Complete Anterior Cruciate Ligament Injury in the Porcine Model}, volume={57}, ISSN={["1938-162X"]}, DOI={10.4085/1062-6050-565-21}, abstractNote={
                  Context
                  Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in female adolescents. Complete ACL tears are typically surgically reconstructed, but few guidelines and very limited data exist regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients.
               }, number={9-10}, journal={JOURNAL OF ATHLETIC TRAINING}, author={Howe, Danielle and Cone, Stephanie G. and Piedrahita, Jorge A. and Spang, Jeffrey T. and Fisher, Matthew B.}, year={2022}, month={Sep}, pages={978–989} }
 @article{sper_proctor_lascina_guo_polkoff_kaeser_simpson_borst_gleason_zhang_et al._2022, title={Allogeneic and xenogeneic lymphoid reconstitution in a RAG2(-/-)IL2RG(y/-) severe combined immunodeficient pig: A preclinical model for intrauterine hematopoietic transplantation}, volume={9}, ISSN={["2297-1769"]}, DOI={10.3389/fvets.2022.965316}, abstractNote={Mice with severe combined immunodeficiency are commonly used as hosts of human cells. Size, longevity, and physiology, however, limit the extent to which immunodeficient mice can model human systems. To address these limitations, we generated RAG2−/−IL2RGy/− immunodeficient pigs and demonstrate successful engraftment of SLA mismatched allogeneic D42 fetal liver cells, tagged with pH2B-eGFP, and human CD34+ hematopoietic stem cells after in utero cell transplantation. Following intrauterine injection at day 42–45 of gestation, fetuses were allowed to gestate to term and analyzed postnatally for the presence of pig (allogeneic) and human (xenogeneic) B cells, T-cells and NK cells in peripheral blood and other lymphoid tissues. Engraftment of allogeneic hematopoietic cells was detected based on co-expression of pH2B-eGFP and various markers of differentiation. Analysis of spleen revealed robust generation and engraftment of pH2B-eGFP mature B cells (and IgH recombination) and mature T-cells (and TCR-β recombination), T helper (CD3+CD4+) and T cytotoxic (CD3+CD8+) cells. The thymus revealed engraftment of pH2B-eGFP double negative precursors (CD4−CD8−) as well as double positive (CD4+, CD8+) precursors and single positive T-cells. After intrauterine administration of human CD34+ hematopoietic stem cells, analysis of peripheral blood and lymphoid tissues revealed the presence of human T-cells (CD3+CD4+ and CD3+CD8+) but no detectable B cells or NK cells. The frequency of human CD45+ cells in the circulation decreased rapidly and were undetectable within 2 weeks of age. The frequency of human CD45+ cells in the spleen also decreased rapidly, becoming undetectable at 3 weeks. In contrast, human CD45+CD3+T-cells comprised >70% of cells in the pig thymus at birth and persisted at the same frequency at 3 weeks. Most human CD3+ cells in the pig's thymus expressed CD4 or CD8, but few cells were double positive (CD4+ CD8+). In addition, human CD3+ cells in the pig thymus contained human T-cell excision circles (TREC), suggesting de novo development. Our data shows that the pig thymus provides a microenvironment conducive to engraftment, survival and development of human T-cells and provide evidence that the developing T-cell compartment can be populated to a significant extent by human cells in large animals.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Sper, Renan B. and Proctor, Jessica and Lascina, Odessa and Guo, Ling and Polkoff, Kathryn and Kaeser, Tobias and Simpson, Sean and Borst, Luke and Gleason, Katherine and Zhang, Xia and et al.}, year={2022}, month={Oct} }
 @article{gonzalez_simon_blondel_fanous_roger_maysonet_devlin_smith_oh_havlik_et al._2022, title={Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing}, volume={13}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-022-33745-4}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, author={Gonzalez, Trevor J. and Simon, Katherine E. and Blondel, Leo O. and Fanous, Marco M. and Roger, Angela L. and Maysonet, Maribel Santiago and Devlin, Garth W. and Smith, Timothy J. and Oh, Daniel K. and Havlik, L. Patrick and et al.}, year={2022}, month={Oct} }
 @article{detwiler_polkoff_gaffney_freytes_piedrahita_2022, title={Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model}, volume={8}, ISSN={["1937-335X"]}, DOI={10.1089/ten.tea.2021.0217}, abstractNote={Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding.}, journal={TISSUE ENGINEERING PART A}, author={Detwiler, Amber and Polkoff, Kathryn and Gaffney, Lewis and Freytes, Donald O. and Piedrahita, Jorge A.}, year={2022}, month={Aug} }
 @article{polkoff_gupta_green_murphy_chung_gleason_simpson_walker_collins_piedrahita_2022, title={LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis}, volume={12}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-022-13056-w}, abstractNote={Abstract}, number={1}, journal={SCIENTIFIC REPORTS}, author={Polkoff, Kathryn M. and Gupta, Nithin K. and Green, Adrian J. and Murphy, Yanet and Chung, Jaewook and Gleason, Katherine L. and Simpson, Sean G. and Walker, Derek M. and Collins, Bruce and Piedrahita, Jorge A.}, year={2022}, month={Jun} }
 @article{chansoria_asif_gupta_piedrahita_shirwaiker_2022, title={Multiscale Anisotropic Tissue Biofabrication via Bulk Acoustic Patterning of Cells and Functional Additives in Hybrid Bioinks}, volume={1}, ISSN={["2192-2659"]}, DOI={10.1002/adhm.202102351}, abstractNote={Abstract}, journal={ADVANCED HEALTHCARE MATERIALS}, author={Chansoria, Parth and Asif, Suleman and Gupta, Nithin and Piedrahita, Jorge and Shirwaiker, Rohan A.}, year={2022}, month={Jan} }
 @article{moatti_li_sivadanam_cai_ranta_piedrahita_cheng_ligler_greenbaum_2022, title={Ontogeny of cellular organization and LGR5 expression in porcine cochlea revealed using tissue clearing and 3D imaging}, volume={25}, ISSN={["2589-0042"]}, DOI={10.1016/j.isci.2022.104695}, abstractNote={Over 11% of the world's population experience hearing loss. Although there are promising studies to restore hearing in rodent models, the size, ontogeny, genetics, and frequency range of hearing of most rodents' cochlea do not match that of humans. The porcine cochlea can bridge this gap as it shares many anatomical, physiological, and genetic similarities with its human counterpart. Here, we provide a detailed methodology to process and image the porcine cochlea in 3D using tissue clearing and light-sheet microscopy. The resulting 3D images can be employed to compare cochleae across different ages and conditions, investigate the ontogeny of cochlear cytoarchitecture, and produce quantitative expression maps of LGR5, a marker of cochlear progenitors in mice. These data reveal that hair cell organization, inner ear morphology, cellular cartography in the organ of Corti, and spatiotemporal expression of LGR5 are dynamic over developmental stages in a pattern not previously documented.}, number={8}, journal={ISCIENCE}, author={Moatti, Adele and Li, Chen and Sivadanam, Sasank and Cai, Yuheng and Ranta, James and Piedrahita, Jorge A. and Cheng, Alan G. and Ligler, Frances S. and Greenbaum, Alon}, year={2022}, month={Aug} }
 @article{vaden_mathews_yoo_williams_harris_secoura_robertson_gleason_reynolds_piedrahita_2022, title={The use of autologous skeletal muscle progenitor cells for adjunctive treatment of presumptive urethral sphincter mechanism incompetence in female dogs}, volume={8}, ISSN={["1939-1676"]}, url={http://dx.doi.org/10.1111/jvim.16505}, DOI={10.1111/jvim.16505}, abstractNote={Abstract}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, publisher={Wiley}, author={Vaden, Shelly L. and Mathews, Kyle G. and Yoo, James and Williams, James Koudy and Harris, Tonya and Secoura, Patty and Robertson, James and Gleason, Katherine L. and Reynolds, Hannah and Piedrahita, Jorge}, year={2022}, month={Aug} }
 @article{chansoria_asif_polkoff_chung_piedrahita_shirwaiker_2021, title={Characterizing the Effects of Synergistic Thermal and Photo-Cross-Linking during Biofabrication on the Structural and Functional Properties of Gelatin Methacryloyl (GeIMA) Hydrogels}, volume={7}, ISSN={["2373-9878"]}, DOI={10.1021/acsbiomaterials.1c00635}, abstractNote={Gelatin methacryloyl (GelMA) hydrogels have emerged as promising and versatile biomaterial matrices with applications spanning drug delivery, disease modeling, and tissue engineering and regenerative medicine. GelMA exhibits reversible thermal cross-linking at temperatures below 37 °C due to the entanglement of constitutive polymeric chains, and subsequent ultraviolet (UV) photo-cross-linking can covalently bind neighboring chains to create irreversibly cross-linked hydrogels. However, how these cross-linking modalities interact and can be modulated during biofabrication to control the structural and functional characteristics of this versatile biomaterial is not well explored yet. Accordingly, this work characterizes the effects of synergistic thermal and photo-cross-linking as a function of GelMA solution temperature and UV photo-cross-linking duration during biofabrication on the hydrogels' stiffness, microstructure, proteolytic degradation, and responses of NIH 3T3 and human adipose-derived stem cells (hASC). Smaller pore size, lower degradation rate, and increased stiffness are reported in hydrogels processed at lower temperature or prolonged UV exposure. In hydrogels with low stiffness, the cells were found to shear the matrix and cluster into microspheroids, while poor cell attachment was noted in high stiffness hydrogels. In hydrogels with moderate stiffness, ones processed at lower temperature demonstrated better shape fidelity and cell proliferation over time. Analysis of gene expression of hASC encapsulated within the hydrogels showed that, while the GelMA matrix assisted in maintenance of stem cell phenotype (CD44), a higher matrix stiffness resulted in higher pro-inflammatory marker (ICAM1) and markers for cell-matrix interaction (ITGA1 and ITGA10). Analysis of constructs with ultrasonically patterned hASC showed that hydrogels processed at higher temperature possessed lower structural fidelity but resulted in more cell elongation and greater anisotropy over time. These findings demonstrate the significant impact of GelMA material formulation and processing conditions on the structural and functional properties of the hydrogels. The understanding of these material-process-structure-function interactions is critical toward optimizing the functional properties of GelMA hydrogels for different targeted applications.}, number={11}, journal={ACS BIOMATERIALS SCIENCE & ENGINEERING}, author={Chansoria, Parth and Asif, Suleman and Polkoff, Kathryn and Chung, Jaewook and Piedrahita, Jorge A. and Shirwaiker, Rohan A.}, year={2021}, month={Nov}, pages={5175–5188} }
 @article{zhang_lanzoni_hani_overi_cardinale_simpson_pitman_allen_yi_wang_et al._2021, title={Patch grafting, strategies for transplantation of organoids into solid organs such as liver}, volume={277}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2021.121067}, abstractNote={Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. Patch grafting strategies were established enabling transplantation of ≥107th organoids/patch of porcine GFP+ biliary tree stem/progenitors into livers of wild type hosts. Grafts consisted of organoids embedded in soft (~100 Pa) hyaluronan hydrogels, both prepared in serum-free Kubota's Medium; placed against target sites; covered with a silk backing impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of the backing to tether grafts with sutures or glue to target sites. Hyaluronan coatings (~200-300 Pa) onto the serosal surface of the graft served to minimize adhesions with neighboring organs. The organ's clearance of hyaluronans enabled restoration of tissue-specific paracrine and systemic signaling, resulting in return of normal hepatic histology, with donor parenchymal cells uniformly integrated amidst host cells and that had differentiated to mature hepatocytes and cholangiocytes. Grafts containing donor mature hepatocytes, partnered with endothelia, and in the same graft biomaterials as for stem/progenitor organoids, did not engraft. Engraftment occurred if porcine liver-derived mesenchymal stem cells (MSCs) were co-transplanted with donor mature cells. RNA-seq analyses revealed that engraftment correlated with expression of matrix-metalloproteinases (MMPs), especially secreted isoforms that were found expressed strongly by organoids, less so by MSCs, and minimally, if at all, by adult cells. Engraftment with patch grafting strategies occurred without evidence of emboli or ectopic cell distribution. It was successful with stem/progenitor organoids or with cells with a source(s) of secreted MMP isoforms and offers significant potential for enabling cell therapies for solid organs.}, journal={BIOMATERIALS}, author={Zhang, Wencheng and Lanzoni, Giacomo and Hani, Homayoun and Overi, Diletta and Cardinale, Vincenzo and Simpson, Sean and Pitman, Wendy and Allen, Amanda and Yi, Xianwen and Wang, Xicheng and et al.}, year={2021}, month={Oct} }
 @article{howe_cone_piedrahita_collins_fordham_griffith_spang_fisher_2021, title={Sex-specific biomechanics and morphology of the anterior cruciate ligament during skeletal growth in a porcine model}, volume={11}, ISSN={["1554-527X"]}, DOI={10.1002/jor.25207}, abstractNote={Abstract}, journal={JOURNAL OF ORTHOPAEDIC RESEARCH}, author={Howe, Danielle and Cone, Stephanie G. and Piedrahita, Jorge A. and Collins, Bruce and Fordham, Lynn A. and Griffith, Emily H. and Spang, Jeffrey T. and Fisher, Matthew B.}, year={2021}, month={Nov} }
 @article{platt_piedrahita_cascalho_2020, title={Clinical xenotransplantation of the heart: At the watershed}, volume={39}, ISSN={["1557-3117"]}, DOI={10.1016/j.healun.2020.06.002}, abstractNote={See Related Article, page 751 See Related Article, page 751 Transplantation has been the preferred treatment for severe failure of the heart and other organs for more than 3 decades. But, for these decades and longer transplantation could be offered to few who could benefit because donated human organs were scarce. The most obvious solution to the scarcity of human hearts and other organs today, 30 years ago and decades before has been to use animals instead of humans as the source of organs, that is xenotransplantation. Yet, if xenotransplantation has been the most obvious solution, it also has seemed the most frustrating, a proverbial carrot on a stick, held just beyond reach by 3 barriers—immunity, physiologic incompatibility, and infection—of which immunity appeared most daunting.1Cascalho M Platt JL The immunological barrier to xenotransplantation.Immunity. 2001; 14: 437-446Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar The past 3 decades brought some dramatic advances in concept and technologies that might address the barriers to xenotransplantation. Yet, these advances were countered at every turn by discovery of new antigens, more incompatibilities and longer lists of zoonotic organisms. But in the current issue of Journal of Heart and Lung Transplantation, and in a previous report in another journal,2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar,3Längin M Mayr T Reichart B et al.Consistent success in life-supporting porcine cardiac xenotransplantation.Nature. 2018; 564: 430-433Crossref PubMed Scopus (142) Google Scholar Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar report > 3-month survival and function of orthotopic pig cardiac xenografts in baboons given clinically acceptable immunosuppression. The results “fulfill for the first time the preclinical efficacy” standard an International Society for Heart and Lung Transplantation (ISHLT) committee proposed as a premise for launching clinical trials in cardiac xenotransplantation.2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar,4Cooper DK Keogh AM Brink J et al.Report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation: the present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases.J Heart Lung Transplant. 2000; 19: 1125-1165Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Did Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar finally capture the carrot and take, what they call “the final step toward clinical xenotransplantation” or did they just advance the stick, with unenumerated barriers keeping clinical application beyond reach? In 2000, an advisory committee of the ISHLT reported on the status of research into the barriers to clinical cardiac and pulmonary xenotransplantation and recommended that “a clinical trial should be considered when approximately 60% survival of life-supporting pig organs in non-human primates for a minimum of 3 months [has been achieved in] at least 10 animals.”4Cooper DK Keogh AM Brink J et al.Report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation: the present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases.J Heart Lung Transplant. 2000; 19: 1125-1165Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar The report culminated a decade of high profile discovery and debate. Key immune, physiologic, and infectious barriers to xenotransplantation had been identified and dramatic advances had been reported in genetic engineering, reproductive cloning, and development of biologic technologies for suppressing immunity and inducing tolerance (see Cascalho and Platt1Cascalho M Platt JL The immunological barrier to xenotransplantation.Immunity. 2001; 14: 437-446Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar for review). But, the exciting advances that made clinical application seem so close also fueled concern about a long-known but little-understood endogenous retrovirus of pigs (porcine endogenous retrovirus) and about novel organisms that might develop in and spread beyond the immunosuppressed recipient. Although the need for organs for transplantation was urgent and increasing the ISHLT committee suggested only "consideration" of clinical trials because the proposed standard was still far from being achieved. Only 1 of numerous heterotopic cardiac xenografts in non-human primates had survived 3 months, and this graft suffered severe immune and inflammatory injury. Thus, the ISHLT committee correctly asserted that as of 2000, the “immune barriers [had] not yet been overcome.”4Cooper DK Keogh AM Brink J et al.Report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation: the present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases.J Heart Lung Transplant. 2000; 19: 1125-1165Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Equally important was that only 1 orthotopic porcine cardiac xenograft had as yet supported the life of a non-human primate treated with clinically acceptable immunosuppression regimen and that for 39 days.5Vial CM Ostlie DJ Bhatti FN et al.Life supporting function for over one month of a transgenic porcine heart in a baboon.J Heart Lung Transplant. 2000; 19: 224-229Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar In 2016, the prospects for clinical application of xenotransplantation changed dramatically. Mohiuddin et al6Mohiuddin MM Singh AK Corcoran PC et al.Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.Nat Commun. 2016; 7: 11138Crossref PubMed Scopus (190) Google Scholar reported that heterotopic porcine cardiac xenografts expressing a human complement regulator (CD46) and human thrombomodulin and lacking Galα1-3Gal (owing targeting of α1,3-galactosyltransferase) could survive more than a year (up to 945 days) in baboons treated with a clinically-acceptable regimen that included anti-rhesus monkey CD40. Because the xenografts were heterotopic, the potential for providing physiologic support remained uncertain but this success in overcoming immune barriers reinvigorated discussion about whether and how xenotransplantation might be applied in patients. In 2018, Längin et al3Längin M Mayr T Reichart B et al.Consistent success in life-supporting porcine cardiac xenotransplantation.Nature. 2018; 564: 430-433Crossref PubMed Scopus (142) Google Scholar reported 4 successful orthotopic porcine xenografts in baboons that functioned until the recipients were euthanized, per protocol on days 90, 90, 182, and 195. The xenografts were from pigs with the same or similar genetic modifications as those used by Mohiuddin et al,6Mohiuddin MM Singh AK Corcoran PC et al.Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.Nat Commun. 2016; 7: 11138Crossref PubMed Scopus (190) Google Scholar and the baboons were treated with immunosuppression similar to that of Mohiuddin et al6Mohiuddin MM Singh AK Corcoran PC et al.Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.Nat Commun. 2016; 7: 11138Crossref PubMed Scopus (190) Google Scholar (plus an mammalian target of rapamycin inhibitor to limit myocardial remodeling7Kushwaha SS Raichlin E Sheinin Y et al.Sirolimus affects cardiomyocytes to reduce left ventricular mass in heart transplant recipients.Eur Heart J. 2008; 29: 2742-2750Crossref PubMed Scopus (41) Google Scholar). The present report2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar adds 4 orthotopic xenografts to the series. The xenografts in 2 recipients exhibited no evidence of significant rejection but systemic complications in the baboons with grafts harboring Cytomegalovirus (porcine cytomegalovirus [pCMV]) necessitated termination on Days 15 and 27. Two other xenografts functioned at 90 days, when the recipients were euthanized per protocol. Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar portray the 6 of 8 orthotopic xenografts functioning 3 months or longer as “well exceeding the key requirement of the ISHLT of the ISHLT International Advisory Board.” Some might questionwhether 6 of 8 xenografts functioning 3 months or more well exceeds or meets the ISHLT standard (60% of at least 10 life-supporting xenografts functioning 3 months4Cooper DK Keogh AM Brink J et al.Report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation: the present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases.J Heart Lung Transplant. 2000; 19: 1125-1165Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We think giving more than passing mention to this standard formulated more than 15 years before the immunologic barriers had been overcome, obscures the significance of Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar’s accomplishment. If a porcine cardiac xenograft can support a non-human primate treated with a clinically acceptable regimen of immunosuppression; then, we must urgently consider what more, if anything, is needed to offer a cardiac xenograft to a dying human patient. And, we must explain why those facing imminent death for want of a transplant cannot be permitted xenotransplantation instead. Yet, after repeatedly claiming to have met or exceeded the standard for clinical application of xenotransplantation,2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar,3Längin M Mayr T Reichart B et al.Consistent success in life-supporting porcine cardiac xenotransplantation.Nature. 2018; 564: 430-433Crossref PubMed Scopus (142) Google Scholar Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar now demurs, saying only that “more experiments will be necessary.” But, the justification for more experiments is unpersuasive. The authors assert more experiments would “address the efficacy of humanized anti-CD40/CD40L,” but orthotopic cardiac xenografts are extraordinarily inefficient, complex, and insensitive system for testing the efficacy of immune modifiers; indeed, the efficacy of anti-CD40/CD40L was established using heterotopic xenografts.6Mohiuddin MM Singh AK Corcoran PC et al.Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.Nat Commun. 2016; 7: 11138Crossref PubMed Scopus (190) Google Scholar Eight orthotopic xenografts are certainly too few to firmly establish the frequency of success and durability of function porcine cardiac xenografts in baboons. But, larger numbers of xenografts in baboons will reveal little more about the efficacy and durability xenografts might have in humans. The ISHLT committee wisely avoided stipulating a standard error for 60% survival or an end-point beyond 90 days because experimental transplants in non-human primates do not model and potentially misrepresent some key variables in clinical cardiac transplantation.8Platt JL Cascalho M Piedrahita JA Xenotransplantation: progress along paths uncertain from models to application.ILAR J. 2018; 59: 286-308Crossref PubMed Scopus (5) Google Scholar That is not to say experimentation in this model or in others should cease. Clinical cardiac xenotransplantation is likely to raise questions and challenges not foreseen today, and an experimental model could prove invaluable for addressing these. There is another perhaps more pressing reason to question what more experiments can accomplish. We suspect porcine cardiac xenografts in non-human primates might overestimate or misrepresent the barriers to clinical xenotransplantation.8Platt JL Cascalho M Piedrahita JA Xenotransplantation: progress along paths uncertain from models to application.ILAR J. 2018; 59: 286-308Crossref PubMed Scopus (5) Google Scholar The xenografts reported by Mohiuddin et al6Mohiuddin MM Singh AK Corcoran PC et al.Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.Nat Commun. 2016; 7: 11138Crossref PubMed Scopus (190) Google Scholar and Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar were from pigs engineered to express human CD46 and human thrombomodulin to better regulate complement and coagulation in recipients. Yet, these and other proteins interact inefficiently with cognatepartners in baboons.9Lawson JH Platt JL Molecular barriers to xenotransplantation.Transplantation. 1996; 62: 303-310Crossref PubMed Scopus (162) Google Scholar Still more important is the possibility that the human proteins might elicit cellular and/or humoral immunity in baboons. Such immunity developing over time, perhaps when immunosuppression decreases to maintenance, might block regulatory functions or initiate injury to the grafts. Perhaps the anti-CD40 antibodies Mohiuddin et al6Mohiuddin MM Singh AK Corcoran PC et al.Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.Nat Commun. 2016; 7: 11138Crossref PubMed Scopus (190) Google Scholar considers essential are needed to disrupt immunity to the human proteins. Since intensity of immunosuppression could limit clinical application of xenotransplantation, it would be ironic if humans were found to need less intense regimens than baboons because humans are tolerant to the products of these transgenes. Of course, clinical trials might reveal higher or different barriers to xenotransplantation in humans than in baboons. But, that possibility has eluded detection in Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar’s model, and it would seem unlikely that more experiments would overcome the deficiency. Nor will the conduct of more porcine xenografts in baboons reveal much about the barrier posed by infection. The microorganisms of pigs known to be capable of infecting humans have been identified almost entirely by investigation of humans and pigs. Indeed, concerns about porcine endogenous retrovirus arose from investigation of human and pig cells in culture and the waning of those concerns from investigation of human contact with pig organs and xenografts.10Paradis K Langford G Long Z et al.Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group.Science. 1999; 285: 1236-1241Crossref PubMed Scopus (631) Google Scholar Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar associate the demise of 2 baboon recipients with the presence of pCMV in in cardiac xenografts, but the paper provides no evidence that pCMV was transmitted to the recipients or directly caused multiorgan failure that forced termination of the experiments. Perhaps a yet unidentified organism harbored by the baboons compromised innate immune control of pCMV. Regardless, pCMV will be excluded from potential sources of xenografts and since pCMV has not been shown to infect humans, Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar’s experience might reflect an instance in which non-human primates misrepresent a biologic barrier. In 2018, Längin et al3Längin M Mayr T Reichart B et al.Consistent success in life-supporting porcine cardiac xenotransplantation.Nature. 2018; 564: 430-433Crossref PubMed Scopus (142) Google Scholar called the first 4 successful orthotopic cardiac xenografts in baboons a milestone. If those xenografts and the 2 successful xenografts now reported2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar are a milestone, it is because they erase serious doubt about whether a porcine heart can survive and function for months in a non-human primate recipient receiving a clinically acceptable (if not yet optimal) immunosuppression regimen. But, the experiments do not prove clinical xenotransplantation will succeed—only clinical xenotransplants can prove that. If this regimen were used successfully in clinical xenotransplants, the clinical xenotransplants would be appropriately called a milestone, if the regimen failed some other term might be used. Reichart et al2Reichart B, Längin M, Radan J, et al. Pig-to-non-human primate heart transplantation: the final step toward clinical xenotransplantation [e-pub ahead of print]?J Heart Lung Transplant, https://doi.org/10.1016/j.healun.2020.05.004. Accessed July 6, 2020.Google Scholar’s report brings into focus an impending watershed in management heart failure. When clinical trials in cardiac xenotransplantation do begin, whether soon or after many more experiments, interest in the multitude of pre-clinical experiments and committee deliberations concerning the feasibility of xenotransplantation will fade. And, the central question will be whether the essential but intrusive immune modifiers and/or the less than optimal genetic background of engineered pigs8Platt JL Cascalho M Piedrahita JA Xenotransplantation: progress along paths uncertain from models to application.ILAR J. 2018; 59: 286-308Crossref PubMed Scopus (5) Google Scholar can be omitted or changed so that xenotransplantation can approach or even surpass the safety and efficacy of allotransplantation and become the preferred treatment for organ failure. That shift in focus as difficult to imagine as it may be will fuel identification and surmounting of barriers unapparent in today's pre-clinical models. If this impending change in interest in xenotransplantation seems fanciful, it just recapitulates how interest in allotransplantation changed 60 years ago. The authors have no conflicts of interest to declare. The work be the authors pertinent to this communication was supported by the National Institutes of Health (AI122369; OD023138) Pig-to-non-human primate heart transplantation: The final step toward clinical xenotransplantation?The Journal of Heart and Lung TransplantationVol. 39Issue 8PreviewThe demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Full-Text PDF}, number={8}, journal={JOURNAL OF HEART AND LUNG TRANSPLANTATION}, author={Platt, Jeffrey L. and Piedrahita, Jorge A. and Cascalho, Marilia}, year={2020}, month={Aug}, pages={758–760} }
 @article{polkoff_chung_simpson_gleason_piedrahita_2020, title={In Vitro Validation of Transgene Expression in Gene-Edited Pias Using CRISPR Transcriptional Activators}, volume={3}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2020.0037}, abstractNote={The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.}, number={5}, journal={CRISPR JOURNAL}, author={Polkoff, Kathryn M. and Chung, Jaewook and Simpson, Sean G. and Gleason, Katherine and Piedrahita, Jorge A.}, year={2020}, month={Oct}, pages={409–418} }
 @article{cone_lambeth_piedrahita_spang_fisher_2020, title={Joint laxity varies in response to partial and complete anterior cruciate ligament injuries throughout skeletal growth}, volume={101}, ISSN={["1873-2380"]}, DOI={10.1016/j.jbiomech.2020.109636}, abstractNote={Anterior cruciate ligament (ACL) injuries are increasingly common in the skeletally immature population. As such there is a need to increase our understanding of the biomechanical function of the joint following partial and complete ACL injury during skeletal growth. In this work, we aimed to assess changes in knee kinematics and loading of the remaining soft tissues following both partial and complete ACL injury in a porcine model. To do so, we applied anterior-posterior tibial loads and varus-valgus moments to stifle joints of female pigs ranging from early juvenile to late adolescent ages and assessed both kinematics and in-situ loads carried in the bundles of the ACL and other soft tissues including the collateral ligaments and the menisci. Partial ACL injury led to increased anterior tibial translation only in late adolescence and small increases in varus-valgus rotation at all ages. Complete ACL injury led to substantial increases in translation and rotation at all ages. At all ages, the medial collateral ligament and the medial meniscus combined to resist the majority of applied anterior tibial load following complete ACL transection. Across all ages and flexion angles, the contribution of the MCL ranged from 45 to 90% of the anterior load and the contribution of the medial meniscus ranged from 14 to 35% of the anterior load. These findings add to our current understanding of age-specific functional properties of both healthy and injured knees during skeletal growth.}, journal={JOURNAL OF BIOMECHANICS}, author={Cone, Stephanie G. and Lambeth, Emily P. and Piedrahita, Jorge A. and Spang, Jeffrey T. and Fisher, Matthew B.}, year={2020}, month={Mar} }
 @article{moatti_cai_li_sattler_edwards_piedrahita_ligler_greenbaum_2020, title={Three-dimensional imaging of intact porcine cochlea using tissue clearing and custom-built light-sheet microscopy}, volume={11}, ISSN={["2156-7085"]}, url={http://dx.doi.org/10.1364/boe.402991}, DOI={10.1364/BOE.402991}, abstractNote={Hearing loss is a prevalent disorder that affects people of all ages. On top of the existing hearing aids and cochlear implants, there is a growing effort to regenerate functional tissues and restore hearing. However, studying and evaluating these regenerative medicine approaches in a big animal model (e.g. pigs) whose anatomy, physiology, and organ size are similar to a human is challenging. In big animal models, the cochlea is bulky, intricate, and veiled in a dense and craggy otic capsule. These facts complicate 3D microscopic analysis that is vital in the cochlea, where structure-function relation is time and again manifested. To allow 3D imaging of an intact cochlea of newborn and juvenile pigs with a volume up to ∼ 250 mm3, we adapted the BoneClear tissue clearing technique, which renders the bone transparent. The transparent cochleae were then imaged with cellular resolution and in a timely fashion, which prevented bubble formation and tissue degradation, using an adaptive custom-built light-sheet fluorescence microscope. The adaptive light-sheet microscope compensated for deflections of the illumination beam by changing the angles of the beam and translating the detection objective while acquiring images. Using this combination of techniques, macroscopic and microscopic properties of the cochlea were extracted, including the density of hair cells, frequency maps, and lower frequency limits. Consequently, the proposed platform could support the growing effort to regenerate cochlear tissues and assist with basic research to advance cures for hearing impairments.}, number={11}, journal={BIOMEDICAL OPTICS EXPRESS}, publisher={The Optical Society}, author={Moatti, Adele and Cai, Yuheng and Li, Chen and Sattler, Tyler and Edwards, Laura and Piedrahita, Jorge and Ligler, Frances S. and Greenbaum, Alon}, year={2020}, month={Nov}, pages={6181–6196} }
 @article{gupta_polkoff_qiao_cheng_piedrahita_2019, title={200 Developing exosomes as a mediator for CRISPR/Cas-9 delivery}, volume={31}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDV31N1AB200}, DOI={10.1071/RDV31N1AB200}, abstractNote={
CRISPR/Cas systems present a powerful gene-editing tool with the potential for widespread therapeutic use; however, current methods of in vivo delivery such as adeno-associated viruses (AAV) may stimulate an immune response, creating the need for an alternative for delivery of CRISPR/Cas9. Exosomes are small vesicles that are released by cells and serve as a delivery system for RNA, proteins, and various molecules to other cells. The focus of this project was to use exosomes as a delivery system for Cas9, exploiting their high uptake by target cells and their ability to avoid the immune system in vivo. Porcine fetal fibroblasts (PFF) were grown to 80% confluency; after 48h, exosomes were isolated and concentrated from conditioned media by filtration with a 0.22-μm filter followed by 100-kDa molecular weight cutoff filter. Transmission electron microscopy, Western blotting for presence of CD81, and an uptake assay for exosomes stained with the lipophilic dye DiI (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were used to characterise isolated exosomes, and average particle size was evaluated by NanoSight (Salisbury, United Kingdom). After characterisation, exosomes were loaded with Cas9 (PNA Bio, Newbury Park, CA, USA) using sonication, incubation with saponin, or extrusion. For each method of loading, 1.0×1011 exosomes and 500ng of Cas9 were used. For sonication, exosomes and Cas9 were sonicated 4 times: 4s on/2s off, left on ice for 2min, and then repeated for 4 more cycles. Loaded exosomes were then incubated at 37°C for 20min. For incubation with saponin, 100μL of 0.6% saponin solution was made in PBS, mixed with exosomes and Cas9, and then incubated on a shaker at 800 rpm for 20min. For extrusion, exosomes and Cas9 were extruded (Avanti Polar Lipids, Alabaster, AL, USA) 10, 15, or 20 times through a 0.22-μm filter. To evaluate efficiency of Cas9 loading into exosomes, loaded exosome samples were split in half, with one-half receiving a proteinase K digest (100μg mL−1) to remove free Cas9 and the other receiving no treatment. Proteinase K-treated and untreated samples were then compared side by side on Western blot staining for Cas9. ImageJ software (National Institutes for Health, Bethesda, MD, USA) was used to quantify band intensity and loading efficiency. With optimal conditions, our preliminary results show loading efficiency for sonication and saponin to be 16.7 and 19.2%, respectively, whereas loading by extrusion was undetectable. For CRISPR/Cas targeting, transgenic PFF carrying one copy of H2B-GFP were used to test delivery of ribonucleotide protein complex (RNP). To verify efficiency of the guide (g)RNA targeting green fluorescent protein (GFP), cells were nucleofected with Cas9 and gRNA. The DNA was extracted, PCR amplified, and sequenced (Eton Bioscience, San Diego, CA, USA) and then evaluated for indels with TIDE, resulting in a 53.2% cleavage efficiency. Next, exosomes will be loaded with RNP to knockout GFP in H2B-GFP cells, and targeting efficiency will be evaluated by flow cytometry and TIDE. We hypothesise that based on loading efficiency and target cell uptake, exosomes will present a safe and efficient method for in vitro and in vivo delivery of Cas9.
The financial support of the Comparative Medicine Institute is gratefully acknowledged.
}, number={1}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Gupta, N. and Polkoff, K. and Qiao, L. and Cheng, K. and Piedrahita, J.}, year={2019}, pages={225} }
 @article{cone_lambeth_ru_fordham_piedrahita_spang_fisher_2019, title={Biomechanical Function and Size of the Anteromedial and Posterolateral Bundles of the ACL Change Differently with Skeletal Growth in the Pig Model}, volume={477}, ISSN={["1528-1132"]}, DOI={10.1097/CORR.0000000000000884}, abstractNote={ACL injuries are becoming increasingly common in children and adolescents, but little is known regarding age-specific ACL function in these patients. To improve our understanding of changes in musculoskeletal tissues during growth and given the limited availability of pediatric human cadaveric specimens, tissue structure and function can be assessed in large animal models, such as the pig.Using cadaveric porcine specimens ranging throughout skeletal growth, we aimed to assess age-dependent changes in (1) joint kinematics under applied AP loads and varus-valgus moments, (2) biomechanical function of the ACL under the same loads, (3) the relative biomechanical function of the anteromedial and posterolateral bundles of the ACL; and (4) size and orientation of the anteromedial and posterolateral bundles.Stifle joints (analogous to the human knee) were collected from female Yorkshire crossbreed pigs at five ages ranging from early youth to late adolescence (1.5, 3, 4.5, 6, and 18 months; n = 6 pigs per age group, 30 total), and MRIs were performed. A robotic testing system was used to determine joint kinematics (AP tibial translation and varus-valgus rotation) and in situ forces in the ACL and its bundles in response to applied anterior tibial loads and varus-valgus moments. To see if morphological changes to the ACL compared with biomechanical changes, ACL and bundle cross-sectional area, length, and orientation were calculated from MR images.Joint kinematics decreased with increasing age. Normalized AP tibial translation decreased by 44% from 1.5 months (0.34 ± 0.08) to 18 months (0.19 ± 0.02) at 60° of flexion (p < 0.001) and varus-valgus rotation decreased from 25° ± 2° at 1.5 months to 6° ± 2° at 18 months (p < 0.001). The ACL provided the majority of the resistance to anterior tibial loading at all age groups (75% to 111% of the applied anterior force; p = 0.630 between ages). Anteromedial and posterolateral bundle function in response to anterior loading and varus torque were similar in pigs of young ages. During adolescence (4.5 to 18 months), the in situ force carried by the anteromedial bundle increased relative to that carried by the posterolateral bundle, shifting from 59% ± 22% at 4.5 months to 92% ± 12% at 18 months (data for 60° of flexion, p < 0.001 between 4.5 and 18 months). The cross-sectional area of the anteromedial bundle increased by 30 mm throughout growth from 1.5 months (5 ± 2 mm) through 18 months (35 ± 8 mm; p < 0.001 between 1.5 and 18 months), while the cross-sectional area of the posterolateral bundle increased by 12 mm from 1.5 months (7 ± 2 mm) to 4.5 months (19 ± 5 mm; p = 0.004 between 1.5 and 4.5 months), with no further growth (17 ± 7 mm at 18 months; p = 0.999 between 4.5 and 18 months). However, changes in length and orientation were similar between the bundles.We showed that the stifle joint (knee equivalent) in the pig has greater translational and rotational laxity in early youth (1.5 to 3 months) compared with adolescence (4.5 to 18 months), that the ACL functions as a primary stabilizer throughout growth, and that the relative biomechanical function and size of the anteromedial and posterolateral bundles change differently with growth.Given the large effects observed here, the age- and bundle-specific function, size, and orientation of the ACL may need to be considered regarding surgical timing, graft selection, and graft placement. In addition, the findings of this study will be used to motivate pre-clinical studies on the impact of partial and complete ACL injuries during skeletal growth.}, number={9}, journal={CLINICAL ORTHOPAEDICS AND RELATED RESEARCH}, author={Cone, Stephanie G. and Lambeth, Emily P. and Ru, Hongyu and Fordham, Lynn A. and Piedrahita, Jorge A. and Spang, Jeffrey T. and Fisher, Matthew B.}, year={2019}, month={Sep}, pages={2161–2174} }
 @article{cone_piedrahita_spang_fisher_2019, title={In Situ Joint Stiffness Increases During Skeletal Growth but Decreases Following Partial and Complete Anterior Cruciate Ligament Injury}, volume={141}, ISSN={["1528-8951"]}, DOI={10.1115/1.4044582}, abstractNote={Abstract}, number={12}, journal={JOURNAL OF BIOMECHANICAL ENGINEERING-TRANSACTIONS OF THE ASME}, author={Cone, Stephanie G. and Piedrahita, Jorge A. and Spang, Jeffrey T. and Fisher, Matthew B.}, year={2019}, month={Dec} }
 @article{cone_piercy_lambeth_ru_piedrahita_spang_fordham_fisher_2019, title={Tissue-specific changes in size and shape of the ligaments and tendons of the porcine knee during post-natal growth}, volume={14}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0219637}, abstractNote={Prior studies have analyzed growth of musculoskeletal tissues between species or across body segments; however, little research has assessed the differences in similar tissues within a single joint. Here we studied changes in the length and cross-sectional area of four ligaments and tendons, (anterior cruciate ligament, patellar tendon, medial collateral ligament, lateral collateral ligament) in the tibiofemoral joint of female Yorkshire pigs through high-field magnetic resonance imaging throughout growth. Tissue lengths increased by 4-to 5-fold from birth to late adolescence across the tissues while tissue cross-sectional area increased by 10-20-fold. The anterior cruciate ligament and lateral collateral ligament showed allometric growth favoring change in length over change in cross-sectional area while the patellar tendon and medial collateral ligament grow in an isometric manner. Additionally, changes in the length and cross-sectional area of the anterior cruciate ligament did not increase as much as in the other ligaments and tendon of interest. Overall, these findings suggest that musculoskeletal soft tissue morphometry can vary within tissues of similar structure and within a single joint during post-natal growth.}, number={10}, journal={PLOS ONE}, author={Cone, Stephanie G. and Piercy, Hope E. and Lambeth, Emily P. and Ru, Hongyu and Piedrahita, Jorge A. and Spang, Jeffrey T. and Fordham, Lynn A. and Fisher, Matthew B.}, year={2019}, month={Oct} }
 @article{chung_zhang_collins_sper_gleason_simpson_koh_sommer_flowers_petters_et al._2018, title={High mobility group A2 (HMGA2) deficiency in pigs leads to dwarfism, abnormal fetal resource allocation, and cryptorchidism}, volume={115}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1721630115}, DOI={10.1073/pnas.1721630115}, abstractNote={Significance}, number={21}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Chung, Jaewook and Zhang, Xia and Collins, Bruce and Sper, Renan B. and Gleason, Katherine and Simpson, Sean and Koh, Sehwon and Sommer, Jeffrey and Flowers, William L. and Petters, Robert M. and et al.}, year={2018}, month={May}, pages={5420–5425} }
 @article{jungersen_piedrahita_2018, title={INTRODUCTION: Immune Relevant Animal Models: Opportunities and Challenges}, volume={59}, ISSN={["1930-6180"]}, DOI={10.1093/ilar/ilz014}, abstractNote={Abstract}, number={3}, journal={ILAR JOURNAL}, author={Jungersen, Gregers and Piedrahita, Jorge}, year={2018}, pages={209–210} }
 @misc{platt_cascalho_piedrahita_2018, title={Xenotransplantation: Progress Along Paths Uncertain from Models to Application}, volume={59}, ISSN={["1930-6180"]}, DOI={10.1093/ilar/ily015}, abstractNote={Abstract}, number={3}, journal={ILAR JOURNAL}, author={Platt, Jeffrey L. and Cascalho, Marilia and Piedrahita, Jorge A.}, year={2018}, pages={286–308} }
 @article{piedrahita_williams_2017, title={Animal Models in Tissue Engineering. Part I}, volume={23}, ISSN={1937-3384 1937-3392}, url={http://dx.doi.org/10.1089/ten.TEC.2017.0402}, DOI={10.1089/ten.tec.2017.0402}, abstractNote={Animal models play a central and pivotal role in tissue engineering. Although advances in areas such as 3D printing and bioreactor technologies now permit the in vitro development and testing of complex scaffold/cell composites, in vivo testing remains critical not only for refining methods being developed but also for the critical efficacy and safety testing required for regulatory approval. Yet, choosing the appropriate model for a particular application remains a challenge, as each model has its own strengths and weaknesses. In some cases, there are size issues to contend with as scale-up of a 3D structure brings with it considerable challenges with regard to diffusion, infiltration, and structural forces. In others, physiological differences between species make selection of the appropriate animal model that best represents the human disease or injury critical.}, number={11}, journal={Tissue Engineering Part C: Methods}, publisher={Mary Ann Liebert Inc}, author={Piedrahita, Jorge A. and Williams, J. Koudy}, year={2017}, month={Nov}, pages={641–642} }
 @article{piedrahita_williams_2017, title={Animal Models in Tissue Engineering. Part II}, volume={23}, ISSN={1937-3384 1937-3392}, url={http://dx.doi.org/10.1089/ten.TEC.2017.0458}, DOI={10.1089/ten.TEC.2017.0458}, abstractNote={Animal models play a central and pivotal role in tissue engineering. Although advances in areas such as 3D printing and bioreactor technologies now permit the in vitro development and testing of complex scaffold/cell composites, in vivo testing remains critical not only for refining methods being developed but also for the critical efficacy and safety testing required for regulatory approval. Yet, choosing the appropriate model for a particular application remains a challenge, as each model has its own strengths and weaknesses. In some cases, there are size issues to contend with as scale-up of a 3D structure brings with it considerable challenges with regard to diffusion, infiltration, and structural forces. In others, physiological differences between species make selection of the appropriate animal model that best represents the human disease or injury critical.}, number={12}, journal={Tissue Engineering Part C: Methods}, publisher={Mary Ann Liebert Inc}, author={Piedrahita, Jorge A. and Williams, J. Koudy}, year={2017}, month={Dec}, pages={827–828} }
 @article{gonçalves_bressan_roballo_meirelles_xavier_fukumasu_williams_breen_koh_sper_et al._2017, title={Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts}, volume={92}, ISSN={0093-691X}, url={http://dx.doi.org/10.1016/j.theriogenology.2017.01.013}, DOI={10.1016/j.theriogenology.2017.01.013}, abstractNote={Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.}, journal={Theriogenology}, publisher={Elsevier BV}, author={Gonçalves, N.J.N. and Bressan, F.F. and Roballo, K.C.S. and Meirelles, F.V. and Xavier, P.L.P. and Fukumasu, H. and Williams, C. and Breen, M. and Koh, S. and Sper, R. and et al.}, year={2017}, month={Apr}, pages={75–82} }
 @article{sper_koh_zhang_simpson_collins_sommer_petters_caballero_platt_piedrahita_2017, title={Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies}, volume={12}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0169242}, DOI={10.1371/journal.pone.0169242}, abstractNote={Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model.}, number={1}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Sper, Renan B. and Koh, Sehwon and Zhang, Xia and Simpson, Sean and Collins, Bruce and Sommer, Jeff and Petters, Robert M. and Caballero, Ignacio and Platt, Jeff L. and Piedrahita, Jorge A.}, editor={Polejaeva, IrinaEditor}, year={2017}, month={Jan}, pages={e0169242} }
 @article{koh_purser_wysk_piedrahita_2017, title={Improved Chondrogenic Potential and Proteomic Phenotype of Porcine Chondrocytes Grown in Optimized Culture Conditions}, volume={19}, ISSN={2152-4971 2152-4998}, url={http://dx.doi.org/10.1089/cell.2017.0005}, DOI={10.1089/cell.2017.0005}, abstractNote={For successful cartilage tissue engineering, the ability to generate a high number of chondrocytes in vitro while avoiding terminal differentiation or de-differentiation is critical. The ability to accomplish this by using the abundant and easily sampled costal cartilage could provide a practical alternative to the use of articular cartilage and mesenchymal stem cells. Chondrocytes isolated from pig costal cartilage were expanded in either serum-free medium with FGF2 (SFM) or fetal bovine serum-containing medium (SCM), under either high (21%) or low (5%) oxygen conditions. Overall, chondrocytes cultured in SFM and low oxygen (Low-SFM) demonstrated the highest cell growth rate (p < 0.05). The effect of passage number on the differentiation status of the chondrocytes was analyzed by alkaline phosphatase (AP) staining and real-time PCR for known chondrocyte quality markers. AP staining indicated that chondrocytes grown in SCM had a higher proportion of terminally differentiated (hypertrophic) chondrocytes (p < 0.05). At the mRNA level, expression ratios of ACAN/VCAN and COL2/COL1 were significantly higher (p < 0.05) in cells expanded in Low-SFM, indicating reduced de-differentiation. In vitro re-differentiation capacity was assessed after a 6-week induction, and chondrocytes grown in Low-SFM showed similar expression ratios of COL2/COL1 and ACAN/VCAN to native cartilage. Proteomic analysis of in vitro produced cartilage indicated that the Low-SFM condition most closely matched the proteomic profile of native costal and articular cartilage. In conclusion, Low-SFM culture conditions resulted in improved cell growth rates, reduced levels of de-differentiation during expansion, greater ability to re-differentiate into cartilage on induction, and an improved proteomic profile that resembles that of in vivo cartilage.}, number={4}, journal={Cellular Reprogramming}, publisher={Mary Ann Liebert Inc}, author={Koh, Sehwon and Purser, Molly and Wysk, Richard and Piedrahita, Jorge A.}, year={2017}, month={Aug}, pages={232–244} }
 @article{cone_simpson_piedrahita_fordham_spang_fisher_2017, title={Orientation changes in the cruciate ligaments of the knee during skeletal growth: A porcine model}, volume={35}, ISSN={0736-0266}, url={http://dx.doi.org/10.1002/jor.23594}, DOI={10.1002/jor.23594}, abstractNote={ABSTRACT}, number={12}, journal={Journal of Orthopaedic Research}, publisher={Wiley}, author={Cone, Stephanie G. and Simpson, Sean G. and Piedrahita, Jorge A. and Fordham, Lynn A. and Spang, Jeffrey T. and Fisher, Matthew B.}, year={2017}, month={May}, pages={2725–2732} }
 @article{sper_simpson_zhang_collins_piedrahita_2016, title={1 GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMIC STUDIES}, volume={28}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDV28N2AB1}, DOI={10.1071/RDV28N2AB1}, abstractNote={
Transgenic pigs are an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic, and physiological similarities with humans. The development of a transgenic murine model with a fusion of green fluorescent protein (GFP) to histone 2B protein (H2B, protein of nucleosome core) resulted in an easier and more convenient method for tracking cell migration and engraftment levels after transplantation as well as a way to better understand the complexity of molecular regulation within cell cycle/division, cancer biology, and chromosome dynamics. Up to now the development of a stable transgenic large animal model expressing H2B-GFP has not been described. Our objective was to develop the first transgenic porcine H2B-GFP model via CRISPR-CAS9 mediated recombination and somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were cotransfected with CRISPR-CAS9 designed to target the 3′ untranslated region of ACTB locus and a targeting vector containing 1Kb homology arms to ACTB flanking an IRES-H2B-GFP transgene. Four days after transfection GFP cells were fluorescence activated cell sorted. Single cell colonies were generated and analysed by PCR, and heterozygous colonies were used as donor cells for SCNT. The custom designed CRISPR-CAS9 knockin system demonstrated a 2.4% knockin efficiency. From positive cells, 119 SCNT embryos were generated and transferred to a recipient gilt resulting in three positive founder boars (P1 generation). Boars show normal fertility (pregnancies obtained via AI of wild type sows). Generated P1 clones were viable and fertile with a transgene transmission rate of 55.8% (in concordance with Mendel’s law upon chi-square test with P = 0.05). Intranuclear H2B-GFP expression was confirmed via fluorescence microscopy on 8-day in vitro cultured SCNT blastocysts and a variety of tissues (heart, kidney, brain, bladder, skeletal muscle, stomach, skin, and so on) and primary cultured cells (chondrocytes, bone marrow derived, adipocyte derived, neural stem cells, and so on) from P1 cloned boars and F1 42-day fetuses and viable piglets. In addition, chromosome segregation could be easily identified during cell cycle division in in vitro cultured stem cells. Custom designed CRISPR-CAS 9 are able to drive homologous recombination in the ACTB locus in porcine fetal fibroblasts, allowing the generation of the first described viable H2B-GFP porcine model via SCNT. Generated clones and F1 generation expressed H2B-GFP ubiquitously, and transgene transmission rates were with concordance of Mendel’s law. This novel large animal model represents an improved platform for regenerative medicine and chromosome dynamic and cancer biology studies.}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Sper, R. and Simpson, S. and Zhang, X. and Collins, B. and Piedrahita, J.}, year={2016}, pages={130} }
 @article{simpson_gonzalez_chung_blikslager_magness_piedrahita_2016, title={27 AN IMPROVED LARGE ANIMAL MODEL FOR THE STUDY OF ADULT STEM CELLS}, volume={28}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDV28N2AB27}, DOI={10.1071/RDV28N2AB27}, abstractNote={

Murine models for the study of adult stem cell populations have broadened the understanding of previously uncharacterized stem cell niches. The development of murine reporter lines for the leucine-rich repeat-containing G-protein-coupled receptor-5 (Lgr5) has highlighted the importance of this gene as a stem cell marker in the stomach, intestine, hair follicle, liver, and kidney in mice. These models however have significant limitations in terms of translational applications because of anatomical and physiological differences between humans and mice. In order to overcome these limitations, we have sought to develop a porcine LGR5 reporter line. We report the generation of a porcine stem cell reporter line using the combination of transcription activator-like effector nucleases and somatic cell NT. Transcription activator-like effector nuclease-mediated homologous recombination was used to drive the integration of an internal ribosome entry site green fluorescent protein fusion into the 3′ untranslated region of the LGR5 locus in porcine fetal fibroblast cells. Multiple cell lines were developed and screened for the proper integration event. Upon confirmation of proper integration by genomic DNA sequencing, these lines were used as donors for somatic cell NT. Transfer of the somatic cell NT reconstructed embryos to a surrogate gilt resulted in 3 live births, and the establishment of a founder line of LGR5-green fluorescent protein reporter pigs. We have begun to characterise these lines, having observed fluorescent labelling of putative stem cell populations in the intestinal crypts and hair follicles from these animals. Many of these observations parallel the expression patterns observed in similar murine models. We have confirmed the fluorescent reporter signal by immunohistochemistry using an anti-green fluorescent protein antibody, and are working towards colocalization studies using anti-LGR5 antibodies and RNA in situ hybridization, as well as the characterisation of additional stem cell populations in the pig. The development of this line of transgenic pigs represents significant progress toward the study of adult stem cells, their progenitors, and the stem cell niche, using a large animal model with an anatomy, physiology, and ability to recapitulate human disease that overcomes the current limitations of rodent models.
Funding was provided by NIH R21OD019738.
}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Simpson, S. and Gonzalez, L. and Chung, J. and Blikslager, A. and Magness, S. and Piedrahita, J.}, year={2016}, pages={143} }
 @article{jeong_nelson_niedziela_dickey_2016, title={Effect of Plant Species, Fertilizer Acidity/Basicity, and Fertilizer Concentration on pH of Soilless Root Substrate}, volume={51}, ISSN={["2327-9834"]}, DOI={10.21273/hortsci11237-16}, abstractNote={The objective of this study was to determine how plant species, fertilizer potential acidity/basicity rating (PABR), and fertilizer concentration affect root substrate pH. Three experiments were conducted. In the first experiment, 13 herbaceous species were grown in a root substrate of three sphagnum peatmoss: one perlite (v/v) with deionized water and a neutral fertilizer (NF) with a PABR of 0 for 78 days to determine species relationships to substrate pH. The decrease in substrate pH ranged from 0.14 to 2.45 units, depending on species. In the second experiment, four of the 13 species from the previous trial representing the range of pH suppression were grown under similar growth conditions as the first experiment for 70 days. Substrate pH was lowered in the range of 0.47 to 2.72 units. In the third experiment, three fertilizers with PABRs of 150 kg·t−1 CaCO3 equivalent alkalinity, 0 neutral, and 193 kg·t−1 CaCO3 equivalent acidity were applied in a factorial design at 100 and 200 mg·L−1 N at each irrigation to kalanchoe (the species with the greatest pH suppression from the previous experiments) for 56 days. When applied at the lower fertilizer rate (100 mg·L−1 N), the PABRs resulted in the final substrate pH levels of 4.68, 5.60, and 6.11 for the acidic fertilizer (AF), NF, and basic fertilizer (BF), respectively. At the high fertilizer rate (200 mg·L−1 N), substrate pH declined continuously to 3.97, 4.03, and 4.92 for the AF, NF, and BF, respectively. Expression of PABR depended on the balance between the abiotic (chemical) effect of the fertilizers vs. the biotic (physiological) effects of the fertilizers on microbes and plants. The PABR was best expressed when the fertilizer supply was just adequate or lower indicating a closer connection to the biotic effect.}, number={12}, journal={HORTSCIENCE}, author={Jeong, Ka Yeon and Nelson, Paul V. and Niedziela, Carl E., Jr. and Dickey, David A.}, year={2016}, month={Dec}, pages={1596–1601} }
 @article{hensley_andrade_keene_meurs_tang_wang_caranasos_piedrahita_li_cheng_et al._2015, title={Cardiac regenerative potential of cardiosphere-derived cells from adult dog hearts}, volume={19}, ISSN={1582-1838}, url={http://dx.doi.org/10.1111/jcmm.12585}, DOI={10.1111/jcmm.12585}, abstractNote={Abstract}, number={8}, journal={Journal of Cellular and Molecular Medicine}, publisher={Wiley}, author={Hensley, M. T. and Andrade, J. and Keene, B. and Meurs, Kathryn and Tang, J. N. and Wang, Z. G. and Caranasos, T. G. and Piedrahita, J. and Li, T. S. and Cheng, K. and et al.}, year={2015}, month={Apr}, pages={1805–1813} }
 @article{oliveira_mançanares_oliveira_gonçalves_miglino_perecin_meirelles_piedrahita_ambrósio_2015, title={Characterization of putative haematopoietic cells from bovine yolk sac}, volume={11}, ISSN={1932-6254}, url={http://dx.doi.org/10.1002/TERM.2016}, DOI={10.1002/TERM.2016}, abstractNote={The yolk sac is an extra‐embryonic membrane that plays an important role in early embryonic survival. It is the production site for blood cells during embryonic mammalian development and is a likely source of stem cells. The aim of this study was to identify and characterize the putative haematopoietic cells from the yolk sac of bovine embryos at different stages of gestation. The yolk sac regresses according to gestational age and embryos are characterized into groups (I–V) according to the crown–rump measurement. Groups I–III survived in culture longer and exhibited the formation of cell clusters, whereas groups IV and V could not be maintained in culture for an extended period of time. Flow‐cytometry analysis revealed that groups I–III had similar characteristics, including high expression levels of the haematopoietic markers CD34, CD90 and CD117. In groups IV and V, decreases were observed in the expression levels of CD117 and CD34. Cells were found to be capable of survival post‐cryopreservation and exhibited varying abilities to form colonies in a methylcellulose matrix, depending on gestational age. Cytological analysis revealed the presence of blood cells (lymphocytes and monocytes). Quantitative PCR analysis demonstrated the presence of the haematopoietic progenitor genes GATA3 and LMO2, but not RUNX1. Thus, we have successfully isolated and characterized haematopoietic cells from the bovine embryo yolk sac at varying gestational ages. This study is crucial for the understanding of the development of the haematopoietic system and the embryonic function of this organ. Copyright © 2015 John Wiley & Sons, Ltd.}, number={4}, journal={Journal of Tissue Engineering and Regenerative Medicine}, publisher={Wiley}, author={Oliveira, Vanessa C. and Mançanares, Celina A. F. and Oliveira, Lilian J. and Gonçalves, Natalia J. N. and Miglino, Maria A. and Perecin, Felipe and Meirelles, Flávio V. and Piedrahita, Jorge and Ambrósio, Carlos E.}, year={2015}, month={Feb}, pages={1132–1140} }
 @inbook{koh_piedrahita_2015, title={Generation of Induced Pluripotent Stem Cells (iPSCs) from Adult Canine Fibroblasts}, ISBN={9781493928477 9781493928484}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-4939-2848-4_7}, DOI={10.1007/978-1-4939-2848-4_7}, abstractNote={Induced pluripotent stem cells hold great potential in regenerative medicine as it enables to generate pluripotent stem cells from any available cell types. Ectopic expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) can reprogram fibroblasts directly to pluripotency as shown in multiple species. Here, we describe detailed protocols for generation of iPSCs from adult canine fibroblasts. Robust canine iPSCs will provide powerful tools not only to study human diseases, but also for the development of therapeutic approaches.}, booktitle={Methods in Molecular Biology}, publisher={Springer New York}, author={Koh, Sehwon and Piedrahita, Jorge A.}, year={2015}, pages={69–78} }
 @article{vandergriff_de andrade_tang_hensley_piedrahita_caranasos_cheng_2015, title={Intravenous Cardiac Stem Cell-Derived Exosomes Ameliorate Cardiac Dysfunction in Doxorubicin Induced Dilated Cardiomyopathy}, volume={2015}, ISSN={1687-966X 1687-9678}, url={http://dx.doi.org/10.1155/2015/960926}, DOI={10.1155/2015/960926}, abstractNote={Despite the efficacy of cardiac stem cells (CSCs) for treatment of cardiomyopathies, there are many limitations to stem cell therapies. CSC-derived exosomes (CSC-XOs) have been shown to be responsible for a large portion of the regenerative effects of CSCs. Using a mouse model of doxorubicin induced dilated cardiomyopathy, we study the effects of systemic delivery of human CSC-XOs in mice. Mice receiving CSC-XOs showed improved heart function via echocardiography, as well as decreased apoptosis and fibrosis. In spite of using immunocompetent mice and human CSC-XOs, mice showed no adverse immune reaction. The use of CSC-XOs holds promise for overcoming the limitations of stem cells and improving cardiac therapies.}, journal={Stem Cells International}, publisher={Hindawi Limited}, author={Vandergriff, Adam C. and de Andrade, James Bizetto Meira and Tang, Junnan and Hensley, M. Taylor and Piedrahita, Jorge A. and Caranasos, Thomas G. and Cheng, Ke}, year={2015}, pages={1–8} }
 @article{chung_zhang_colins_howard_simpson_salmon_koh_sper_byrd_piedrahita_2014, title={5 DISRUPTION OF THE HIGH MOBILITY GROUP AT-HOOK 2 (HMGA2) GENE IN SWINE REDUCES POSTNATAL GROWTH}, volume={26}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDV26N1AB5}, DOI={10.1071/RDV26N1AB5}, abstractNote={

The high mobility group AT-hook 2 (HMGA2) protein has been shown to be a crucial gene for cell growth, proliferation, and apoptosis; HMGA2 is also a strong biological candidate for growth, because mutations in this gene alter body size in mice and humans. Compared with wild-type controls, adult mice lacking HMGA2 are 60% smaller, and adult heterozygous mutants are 20% smaller. In humans, HMGA2 has been associated with adult and childhood height without any other deleterious effect. Additionally, a microdeletion in the HMGA2 gene in a human patient resulted in short stature, with no dysmorphologies and normal puberty. In order to determine the effect of HMGA2 on fetal and adult growth in pigs, a transgenic pig line deficient in HMGA2 expression was generated by gene targeting in fetal fibroblasts (FF). Using a targeting vector carrying a reporter gene, and homology arms specific to HMGA2, heterozygous mutant cell lines were generated. The cell lines were then used to generate 6 heterozygous females by somatic cell nuclear transfer (SCNT). Bodyweights and lengths from snout to base of tail were measured every 2 weeks for a year for mutant (n = 6) and wild-type farm gilts (n = 6). Data were analysed by one-way ANOVA. As in mice, disruption of one allele of the HMGA2 gene resulted in 25% reduction in weight (P < 0.0001) and 14% reduction in length (P < 0.0001). Early in postnatal growth (2 months), weights of mutants were not different than wild-type. However, mutants were 20 to 35% lighter (P < 0.05) during mid stages (6 months) and 25 to 30% (P < 0.0001) in late stages (3 months). The same insertional mutation generated 8 heterozygous male clones by SCNT. In addition, 7 nontransgenic males from the same FF line were generated as SCNT controls. Bodyweights and lengths were measured every 2 weeks for 30 weeks for HMGA2 heterozygous mutants (n = 8), control SCNT (n = 7) and wild-type farm boars (n = 5). The weight curve of boars showed similar pattern as for mutant gilts. At 30-week postnatal stage, mutants were 17% (P < 0.05) and 16% (P < 0.05) lighter in weight compared with littermate and wild-type animals, respectively. We are presently developing homozygous HMGA2 mutant lines. Currently, 3 of 6 heterozygous gilts have been bred with heterozygous boars, with 1 confirmed pregnancy. The expectation is that the homozygous animals will, like mice, be 60% smaller than the wild-type animals. The approach described here will result not only in a valuable large-animal model of dwarfism, but also in a tool to reduce the size of existing transgenic and nontransgenic swine lines. This, in turn, will increase the receptivity of valuable transgenic lines by the biomedical community.
Funding for this work was provided by NIH grant R21-OD010553 to JP.
}, number={1}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Chung, J. and Zhang, X. and Colins, B. and Howard, K. and Simpson, S. and Salmon, C. and Koh, S. and Sper, R. and Byrd, C. and Piedrahita, J.}, year={2014}, pages={117} }
 @misc{koh_piedrahita_2014, title={From "ES-like" cells to induced pluripotent stem cells: A historical perspective in domestic animals}, volume={81}, ISSN={["1879-3231"]}, DOI={10.1016/j.theriogenology.2013.09.009}, abstractNote={Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat; however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as “embryonic stem-like” cells owing to their similar morphologic characteristics to mouse ESCs, but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of iPSCs. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of iPSCs.}, number={1}, journal={THERIOGENOLOGY}, author={Koh, Sehwon and Piedrahita, Jorge A.}, year={2014}, month={Jan}, pages={103–111} }
 @article{gonçalves_ambrósio_piedrahita_2014, title={Stem Cells and Regenerative Medicine in Domestic and Companion Animals: A Multispecies Perspective}, volume={49}, ISSN={0936-6768}, url={http://dx.doi.org/10.1111/rda.12392}, DOI={10.1111/rda.12392}, abstractNote={Contents}, journal={Reproduction in Domestic Animals}, publisher={Wiley}, author={Gonçalves, NN and Ambrósio, CE and Piedrahita, JA}, year={2014}, month={Oct}, pages={2–10} }
 @article{lim_mccullen_piedrahita_loboa_olby_2013, title={Alternating Current Electric Fields of Varying Frequencies: Effects on Proliferation and Differentiation of Porcine Neural Progenitor Cells}, volume={15}, ISSN={2152-4971 2152-4998}, url={http://dx.doi.org/10.1089/cell.2013.0001}, DOI={10.1089/cell.2013.0001}, abstractNote={Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.}, number={5}, journal={Cellular Reprogramming}, publisher={Mary Ann Liebert Inc}, author={Lim, Ji-Hey and McCullen, Seth D. and Piedrahita, Jorge A. and Loboa, Elizabeth G. and Olby, Natasha J.}, year={2013}, month={Oct}, pages={405–412} }
 @article{gonzalez_williamson_piedrahita_blikslager_magness_2013, title={Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration}, volume={8}, number={6}, journal={PLoS One}, author={Gonzalez, L. M. and Williamson, I. and Piedrahita, J. A. and Blikslager, A. T. and Magness, S. T.}, year={2013} }
 @article{bischoff_tsai_hardison_motsinger-reif_freking_nonneman_rohrer_piedrahita_2013, title={Differences in X-Chromosome Transcriptional Activity and Cholesterol Metabolism between Placentae from Swine Breeds from Asian and Western Origins}, volume={8}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0055345}, abstractNote={To gain insight into differences in placental physiology between two swine breeds noted for their dissimilar reproductive performance, that is, the Chinese Meishan and white composite (WC), we examined gene expression profiles of placental tissues collected at 25, 45, 65, 85, and 105 days of gestation by microarrays. Using a linear mixed model, a total of 1,595 differentially expressed genes were identified between the two pig breeds using a false-discovery rate q-value ≤0.05. Among these genes, we identified breed-specific isoforms of XIST, a long non-coding RNA responsible X-chromosome dosage compensation in females. Additionally, we explored the interaction of placental gene expression and chromosomal location by DIGMAP and identified three Sus scrofa X chromosomal bands (Xq13, Xq21, Xp11) that represent transcriptionally active clusters that differ between Meishan and WC during placental development. Also, pathway analysis identified fundamental breed differences in placental cholesterol trafficking and its synthesis. Direct measurement of cholesterol confirmed that the cholesterol content was significantly higher in the Meishan versus WC placentae. Taken together, this work identifies key metabolic pathways that differ in the placentae of two swine breeds noted for differences in reproductive prolificacy.}, number={1}, journal={PLOS ONE}, author={Bischoff, Steve R. and Tsai, Shengdar Q. and Hardison, Nicholas E. and Motsinger-Reif, Alison A. and Freking, Bradley A. and Nonneman, Dan J. and Rohrer, Gary A. and Piedrahita, Jorge A.}, year={2013}, month={Jan} }
 @article{guo_tsai_hardison_james_motsinger-reif_thames_stone_deng_piedrahita_2013, title={Differentially expressed microRNAs and affected biological pathways revealed by modulated modularity clustering (MMC) analysis of human preeclamptic and IUGR placentas}, volume={34}, ISSN={0143-4004}, url={http://dx.doi.org/10.1016/j.placenta.2013.04.007}, DOI={10.1016/j.placenta.2013.04.007}, abstractNote={This study focuses on the implementation of modulated modularity clustering (MMC) a new cluster algorithm for the identification of molecular signatures of preeclampsia and intrauterine growth restriction (IUGR), and the identification of affected microRNAs Eighty-six human placentas from normal (40), growth-restricted (27), and preeclamptic (19) term pregnancies were profiled using Illumina Human-6 Beadarrays. MMC was utilized to generate modules based on similarities in placental transcriptome. Gene Set Enrichment Analysis (GSEA) was used to predict affected microRNAs. Expression levels of these candidate microRNAs were investigated in seventy-one human term placentas as follows: control (29); IUGR (26); and preeclampsia (16). MMC identified two modules, one representing IUGR placentas and one representing preeclamptic placentas. 326 differentially expressed genes in the module representing IUGR and 889 differentially expressed genes in a module representing preeclampsia were identified. Functional analysis of molecular signatures associated with IUGR identified P13K/AKT, mTOR, p70S6K, apoptosis and IGF-1 signaling as being affected. Analysis of variance of GSEA-predicted microRNAs indicated that miR-194 was significantly down-regulated both in preeclampsia (p = 0.0001) and IUGR (p = 0.0304), and miR-149 was significantly down-regulated in preeclampsia (p = 0.0168). Implementation of MMC, allowed identification of genes disregulated in IUGR and preeclampsia. The reliability of MMC was validated by comparing to previous linear modeling analysis of preeclamptic placentas. MMC allowed the elucidation of a molecular signature associated with preeclampsia and a subset of IUGR samples. This allowed the identification of genes, pathways, and microRNAs affected in these diseases.}, number={7}, journal={Placenta}, publisher={Elsevier BV}, author={Guo, L. and Tsai, S.Q. and Hardison, N.E. and James, A.H. and Motsinger-Reif, A.A. and Thames, B. and Stone, E.A. and Deng, C. and Piedrahita, J.A.}, year={2013}, month={Jul}, pages={599–605} }
 @article{farmer_farin_piedrahita_bischoff_farin_2013, title={Expression of antisense of insulin-like growth factor-2 receptor RNA non-coding (AIRN) during early gestation in cattle}, volume={138}, ISSN={0378-4320}, url={http://dx.doi.org/10.1016/J.ANIREPROSCI.2013.01.009}, DOI={10.1016/J.ANIREPROSCI.2013.01.009}, abstractNote={The insulin-like growth factor type 2 receptor (IGF2R) regulates fetal growth by removing IGF2 from circulation. In mice, expression of the Igf2r gene is only imprinted after implantation and is associated with expression of the antisense non-coding (nc)RNA, Airn. The objectives of this study were, first, to determine if bovine AIRN was expressed during developmentally important stages of gestation, and second, to determine if expression of bAIRN was affected by method of embryo production. Control reactions confirmed that sequence verified bAIRN PCR amplicons resulted from RNA within the sample and not from genomic DNA contamination. IGF2R mRNA was expressed in all fetal liver samples at Days 35-55 and 70 of gestation as well as in 8 of 9 Day 15 conceptuses, 10 of 10 Day 18 conceptuses, and in all day 7 blastocyst pools. bAIRN was expressed in all samples of fetal liver at Days 35-55 and 70 of gestation. The proportion of conceptuses that expressed bAIRN increased from 1 of 9 at Day 15 of gestation to 8 of 10 at Day 18 of gestation. No bAIRN was expressed in any blastocyst pools. The relative level of bAIRN was greater (P<0.05) in fetal liver from embryos produced in vivo compared to that from embryos produced in vitro. In summary bAIRN was not expressed in blastocyst-stage embryos, was expressed in an increasing proportion of embryos around the time of maternal recognition of pregnancy and was expressed following implantation. Furthermore, relative levels of bAIRN in bovine fetal liver can be altered by method of embryo production.}, number={1-2}, journal={Animal Reproduction Science}, publisher={Elsevier BV}, author={Farmer, W.T. and Farin, P.W. and Piedrahita, J.A. and Bischoff, S.R. and Farin, C.E.}, year={2013}, month={Apr}, pages={64–73} }
 @article{koh_thomas_tsai_bischoff_lim_breen_olby_piedrahita_2013, title={Growth Requirements and Chromosomal Instability of Induced Pluripotent Stem Cells Generated from Adult Canine Fibroblasts}, volume={22}, ISSN={1547-3287 1557-8534}, url={http://dx.doi.org/10.1089/scd.2012.0393}, DOI={10.1089/scd.2012.0393}, abstractNote={In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.}, number={6}, journal={Stem Cells and Development}, publisher={Mary Ann Liebert Inc}, author={Koh, Sehwon and Thomas, Rachael and Tsai, Shengdar and Bischoff, Steve and Lim, Ji-Hey and Breen, Matthew and Olby, Natasha J. and Piedrahita, Jorge A.}, year={2013}, month={Mar}, pages={951–963} }
 @article{gonzalez_blikslager_piedrahita_magness_2013, title={Su1099 A Translational Porcine Model of Intestinal Stem Cells}, volume={144}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(13)61466-3}, DOI={10.1016/S0016-5085(13)61466-3}, number={5}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Gonzalez, Liara and Blikslager, Anthony and Piedrahita, Jorge A. and Magness, Scott T.}, year={2013}, month={May}, pages={S-398} }
 @article{piedrahita_koh_olby_2012, title={GENERATION AND CHARACTERISATION OF INDUCED PLURIPOTENT STEM CELLS (iPSCs) FROM ADULT CANINE FIBROBLASTS}, volume={24}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv24n1Ab244}, DOI={10.1071/RDv24n1Ab244}, abstractNote={
Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Klf4, Sox2 and c-Myc (OKSM). In his presentation we will describe the derivation of iPS cells from adult canine fibroblast by retroviral OSKM transduction. The isolated canine iPS cells were expanded in three different iPS culture media (FGF2, LIF and FGF2 plus LIF) and only the cells cultured in FGF2 plus LIF showed strong AP activity expressed pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28 as well as ES cells-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1). In vitro differentiation by formation of embryoid bodies (EBs) and directed differentiation showed cell derivatives of all three germ layers as confirmed by expression for AFP, CXCR4 and SOX17 (endoderm), desmin (DES), vimentin (VIM), MSX1 and BMP2 (mesoderm) and glial fibrillary acidic protein (GFAP), TUJ1, NCAM and bIII-tubulin (TUBB, ectoderm). In vivo, the putative canine iPS cells formed simple teratomas that expressed markers for all three germ layers. In summary, we were able to derive induced pluripotent cells from adult somatic cells by using four transcription factors. The isolated canine iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF and LIF is still unknown. This work was funded by a grant from the America Kennel Club to JAP.}, number={1}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Piedrahita, Jorge A. and Koh, Sehwon and Olby, Natasha}, year={2012}, pages={285} }
 @inproceedings{koh_tsai_bischoff_thomas_lim_breen_olby_piedrahita_2012, title={Generation and characterization of induced pluripotent stem cells (iPS) from adult canine fibroblasts}, volume={47}, booktitle={Reproduction in Domestic Animals}, author={Koh, S. and Tsai, S. and Bischoff, S. and Thomas, R. and Lim, J. H. and Breen, M. and Olby, N. and Piedrahita, J.}, year={2012}, pages={418–419} }
 @article{lim_koh_olby_piedrahita_mariani_2012, title={Isolation and characterization of neural progenitor cells from adult canine brains}, volume={73}, ISSN={0002-9645}, url={http://dx.doi.org/10.2460/ajvr.73.12.1963}, DOI={10.2460/ajvr.73.12.1963}, abstractNote={Abstract}, number={12}, journal={American Journal of Veterinary Research}, publisher={American Veterinary Medical Association (AVMA)}, author={Lim, Ji-Hey and Koh, Sehwon and Olby, Natasha J. and Piedrahita, Jorge and Mariani, Christopher L.}, year={2012}, month={Dec}, pages={1963–1968} }
 @article{chung_tsai_james_thames_shytle_piedrahita_2012, title={Lack of genomic imprinting of DNA primase, polypeptide 2 (PRIM2) in human term placenta and white blood cells}, volume={7}, ISSN={["1559-2294"]}, DOI={10.4161/epi.19777}, abstractNote={PRIM2, encoding a subunit of primase involved in DNA replication and transcription, is expressed in the placenta and is crucial for mammalian development and growth. Its role in placental function is not well understood. Recently, PRIM2 was reported as imprinted in human white blood cells (WBC). We report here our failure to confirm imprinting of the PRIM2 locus in human placenta or WBC. The discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database.}, number={5}, journal={EPIGENETICS}, author={Chung, Jaewook and Tsai, Shengdar and James, Andra H. and Thames, Betty H. and Shytle, Stephanie and Piedrahita, Jorge A.}, year={2012}, month={May}, pages={429–431} }
 @article{sommer_jackson_simpson_collins_piedrahita_petters_2012, title={Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies}, volume={21}, ISSN={["0962-8819"]}, DOI={10.1007/s11248-011-9542-6}, abstractNote={The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.}, number={2}, journal={TRANSGENIC RESEARCH}, author={Sommer, Jeffrey R. and Jackson, Lauren R. and Simpson, Sean G. and Collins, Edwin B. and Piedrahita, Jorge A. and Petters, Robert M.}, year={2012}, month={Apr}, pages={383–392} }
 @article{sommer_jackson_simpson_collins_piedrahita_petters_2011, title={336 TRANSGENIC Stra8-EYFP PIGS: A MODEL FOR DEVELOPING MALE GERM CELL TECHNOLOGIES}, volume={23}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv23n1Ab336}, DOI={10.1071/RDv23n1Ab336}, abstractNote={

Stimulated by retinoic acid 8 (STRA8) is a protein that is required for meiotic initiation in both male and female gametes in vertebrates. It is also expressed in embryonic germ cells and neonatal male germ cells of mice. The utility of using the Stra8 promoter to recognise and isolate pre-meiotic male germ cells has been reported by others in the mouse. In order to mark germ cells in male pigs, we cloned 1.6 kb of the mouse Stra8 promoter and used it to develop a reporter plasmid using mitochondrial-localised enhanced yellow fluorescent protein (mEYFP). The Stra8-mEYFP transgenic male pigs were produced using somatic cell nuclear transfer. The mEYFP reporter was expressed and easily detectable in the live germ cells of the mature animals and could be observed during tissue culture. The mitochondrial-localised expression of the EYFP reporter was helpful in observing the size and stage of the germ cell. The mEYPF protein was found to be expressed only in the testis of the transgenic pigs using Western blot analysis, whereas endogenous STRA8 protein was also detected in the lung and brain. Fluorescent immunohistochemistry of testicular sections of the transgenic pigs indicated a similar expression pattern to that of the endogenous STRA8 protein. There was an overlap in the expression of the mEYFP and the endogenous STRA8 protein; however, it was observed that the mEYFP protein was present at an earlier stage of spermatogenesis than the STRA8 protein. Immunocytochemistry performed on plated tubules similarly showed varying intensity in expression between the mEYFP transgene and the endogenous STRA8. The difference in the timing of protein expression may be due to the model created or the use of the mouse Stra8 promoter for the expression of mEYFP. Alternatively, the lag in expression between that of the endogenous STRA8 and mEYFP protein may be due to attenuated translation of the Stra8 mRNA. This transgenic model should be useful for the study of reproduction, development, transplantation, biotechnology, and culture of the pig male germ line.

Supported by North Carolina Agricultural Research Service 02234.

}, number={1}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Sommer, J. R. and Jackson, L. and Simpson, S. and Collins, E. B. and Piedrahita, J. and Petters, R. M.}, year={2011}, pages={264} }
 @inproceedings{piedrahita_olby_2011, title={Perspectives on transgenic livestock in agriculture and biomedicine: An update}, volume={23}, number={1}, booktitle={Reproduction, Fertility and Development}, author={Piedrahita, J. A. and Olby, N.}, year={2011}, pages={56–63} }
 @article{piedrahita_olby_2011, title={Perspectives on transgenic livestock in agriculture and biomedicine: an update}, volume={23}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RD10246}, DOI={10.1071/RD10246}, abstractNote={It has been 30 years since the first transgenic mouse was generated and 26 years since the first example of transferring the technology to livestock was published. While there was tremendous optimism in those initial years, with most convinced that genetically modified animals would play a significant role in agricultural production, that has not come to be. So at first sight one could conclude that this technology has, to a large extent, failed. On the contrary, it is believed that it has succeeded beyond our original expectations, and we are now at what is perhaps the most exciting time in the development and implementation of these technologies. The original goals, however, have drastically changed and it is now biomedical applications that are playing a central role in pushing both technical and scientific developments. The combination of advances in somatic cell nuclear transfer, the development of induced pluripotent stem cells and the completion of the sequencing of most livestock genomes ensures a bright and exciting future for this field, not only in livestock but also in companion animal species.}, number={1}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Piedrahita, Jorge A. and Olby, Natasha}, year={2011}, pages={56} }
 @article{sommer_estrada_collins_bedell_alexander_yang_hughes_mir_gilger_grob_et al._2011, title={Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration}, volume={95}, ISSN={0007-1161}, url={http://dx.doi.org/10.1136/bjophthalmol-2011-300417}, DOI={10.1136/bjophthalmol-2011-300417}, abstractNote={Background Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. Methods Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. Results The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. Conclusions These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.}, number={12}, journal={British Journal of Ophthalmology}, publisher={BMJ}, author={Sommer, J. R. and Estrada, J. L. and Collins, E. B. and Bedell, M. and Alexander, C. A. and Yang, Z. and Hughes, G. and Mir, B. and Gilger, B. C. and Grob, S. and et al.}, year={2011}, month={Aug}, pages={1749–1754} }
 @misc{piedrahita_2011, title={The Role of Imprinted Genes in Fetal Growth Abnormalities}, volume={91}, ISSN={["1542-0760"]}, DOI={10.1002/bdra.20795}, abstractNote={Abstract}, number={8}, journal={BIRTH DEFECTS RESEARCH PART A-CLINICAL AND MOLECULAR TERATOLOGY}, author={Piedrahita, Jorge A.}, year={2011}, month={Aug}, pages={682–692} }
 @article{tsai_hardison_james_motsinger-reif_bischoff_thames_piedrahita_2011, title={Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signalling pathways}, volume={32}, ISSN={["1532-3102"]}, DOI={10.1016/j.placenta.2010.11.014}, abstractNote={The placenta plays an important role as a regulator of fetal nutrition and growth throughout development and placental factors contribute to gestational abnormalities such as preeclampsia. This study describes the genome-wide gene expression profiles of a large (n = 60) set of human placentas in order to uncover gene expression patterns associated with preeclampsia. In addition to confirming changes in expression of soluble factors associated with preeclampsia such as sFLT1 (soluble fms-like tyrosine kinase-1), sENG (soluble endoglin), and INHA (inhibin alpha), we also find changes in immune-associated signaling pathways, offering a potential upstream explanation for the shallow trophoblast invasion and inadequate uterine remodeling typically observed in pathogenesis of preeclampsia. Notably, we also find evidence of preeclampsia-associated placental upregulation of sialic acid acetylesterase (SIAE), a gene functionally associated with autoimmune diseases.}, number={2}, journal={PLACENTA}, author={Tsai, S. and Hardison, N. E. and James, A. H. and Motsinger-Reif, A. A. and Bischoff, S. R. and Thames, B. H. and Piedrahita, J. A.}, year={2011}, month={Feb}, pages={175–182} }
 @article{lim_piedrahita_jackson_ghashghaei_olby_2010, title={Development of a Model of Sacrocaudal Spinal Cord Injury in Cloned Yucatan MiniPigs for Cellular Transplantation Research}, volume={12}, ISSN={["2152-4998"]}, DOI={10.1089/cell.2010.0039}, abstractNote={Research into transplantation strategies to treat spinal cord injury (SCI) is frequently performed in rodents, but translation of results to clinical patients can be poor and a large mammalian model of severe SCI is needed. The pig has been considered an optimal model species in which to perform preclinical testing, and the Yucatan minipig can be cloned successfully utilizing somatic cell nuclear transfer (SCNT). However, induction of paralysis in pigs poses significant welfare and nursing challenges. The present study was conducted to determine whether Yucatan SCNT clones could be used to develop an SCI animal model for cellular transplantation research. First, we demonstrated that transection of the sacrocaudal spinal cord in Yucatan SCNT clones produces profound, quantifiable neurological deficits restricted to the tail. We then established that neurospheres could be isolated from brain tissue of green fluorescence protein (GFP) transfected SCNT clones. Finally, we confirmed survival of transplanted GFP-expressing neural stem cells in the SCI lesion and their differentiation into glial and neuronal lineages for up to 4 weeks without immunosuppression. We conclude that this model of sacrocaudal SCI in Yucatan SCNT clones represents a powerful research tool to investigate the effect of cellular transplantation on axonal regeneration and functional recovery.}, number={6}, journal={CELLULAR REPROGRAMMING}, author={Lim, Ji-Hey and Piedrahita, Jorge A. and Jackson, Lauren and Ghashghaei, Troy and Olby, Natasha J.}, year={2010}, month={Dec}, pages={689–697} }
 @article{lim_boozer_mariani_piedrahita_olby_2010, title={Generation and Characterization of Neurospheres from Canine Adipose Tissue-Derived Stromal Cells}, volume={12}, ISSN={["2152-4971"]}, url={http://dx.doi.org/10.1089/cell.2009.0093}, DOI={10.1089/cell.2009.0093}, abstractNote={Adipose tissue-derived stromal cells (ADSCs) have been identified as a powerful stem cell source for cellular transplantation therapy. The dog is increasingly used as a model of human neurological disease; however, few studies have reported induction of canine ADSCs to neural lineages. We characterized canine ADSCs and investigated whether they could be induced to differentiate into neural lineages. Subcutaneous adipose tissue collected from the dorsal epaxial region of adult dogs aged from 1 to 6 years was cultured to produce ADSCs that were then induced to neural lineages. RT-PCR, flow cytometry, and immunocytochemistry were performed to characterize these cell populations. Morphologically fibroblast-like ADSCs were isolated and had similar characteristics to mesenchymal stem cells. Under neurogenic conditions containing basic fibroblast growth factor and epidermal growth factor, ADSCs formed spherical cellular aggregates that resembled neurospheres. RT-PCR confirmed expression of Sox2 and CD90 by these aggregates. Expression of neural stem/progenitor markers (Nestin, Sox2, Vimentin) and neural lineage markers (A2B5, GFAP, Tuj1) was shown on immunocytochemistry. After differentiation, 60% of the cells were Tuj1 positive. In conclusion, we isolated and generated neural progenitor cells from canine ADSCs. ADSCs have potential for future autologous cell transplantation therapy for neurological disorders.}, number={4}, journal={CELLULAR REPROGRAMMING}, author={Lim, Ji-Hey and Boozer, Lindsay and Mariani, Christopher L. and Piedrahita, Jorge A. and Olby, Natasha J.}, year={2010}, month={Aug}, pages={417–425} }
 @article{mccalla-martin_chen_linder_estrada_piedrahita_2010, title={Varying phenotypes in swine versus murine transgenic models constitutively expressing the same human Sonic hedgehog transcriptional activator, K5-HGLI2ΔN}, volume={19}, ISSN={0962-8819 1573-9368}, url={http://dx.doi.org/10.1007/s11248-010-9362-0}, DOI={10.1007/s11248-010-9362-0}, abstractNote={This study was undertaken to characterize the effects of constitutive expression of the hedgehog transcriptional activator, Gli2, in porcine skin. The keratinocyte-specific human transgene, K5-hGli2 Delta N, was used to produce transgenic porcine lines via somatic cell nuclear transfer techniques. In mice, K5-hGli2 Delta N induces epithelial downgrowths resembling basal cell carcinomas. Our porcine model also developed these basal cell carcinoma-like lesions, however gross tumor development was not appreciated. In contrast to the murine model, diffuse epidermal changes as well as susceptibility to cutaneous infections were seen in the swine model. Histologic analysis of transgenic piglets revealed generalized epidermal changes including: epidermal hyperplasia (acanthosis), elongated rete ridges, parakeratotic hyperkeratosis, epidermal neutrophilic infiltration, capillary loop dilation and hypogranulosis. By 2 weeks of age, the transgenic piglets developed erythematic and edematous lesions at high contact epidermal areas and extensor surfaces of distal limb joints. Despite antibiotic treatment, these lesions progressed to a deep bacterial pyoderma and pigs died or were euthanized within weeks of birth. Non-transgenic littermates were phenotypically normal by gross and histological analysis. In summary, constitutive expression of the human hGli2 Delta N in keratinocytes, results in cutaneous changes that have not been reported in the K5-hGli2 Delta N murine model. These findings indicate a need for a multiple species animal model approach in order to better understand the role of Gli2 in mammalian skin.}, number={5}, journal={Transgenic Research}, publisher={Springer Science and Business Media LLC}, author={McCalla-Martin, Amy C. and Chen, Xiaoxin and Linder, Keith E. and Estrada, Jose L. and Piedrahita, Jorge A.}, year={2010}, month={Jan}, pages={869–887} }
 @article{bischoff_tsai_hardison_motsinger-reif_freking_nonneman_rohrer_piedrahita_2009, title={Characterization of Conserved and Nonconserved Imprinted Genes in Swine}, volume={81}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod.109.078139}, abstractNote={To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE, and SLC38A4, tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.}, number={5}, journal={BIOLOGY OF REPRODUCTION}, author={Bischoff, Steve R. and Tsai, Shengdar and Hardison, Nicholas and Motsinger-Reif, Alison A. and Freking, Brad A. and Nonneman, Dan and Rohrer, Gary and Piedrahita, Jorge A.}, year={2009}, month={Nov}, pages={906–920} }
 @article{caballero_piedrahita_2009, title={EVALUATION OF THE SERRATIA MARCESCENS NUCLEASE (NucA) AS A TRANSGENIC CELL ABLATION SYSTEM IN PORCINE}, volume={20}, ISSN={["1532-2378"]}, DOI={10.1080/10495390903048235}, abstractNote={The efficiency of the Serratia marcescens nuclease encoded by the NucA gene, with or without a nuclear localization signal (NLS), and the commonly used diphtheria toxin A (DTA) were compared for their ability to ablate cells in culture. Constructs containing the test genes driven by the β-actin promoter coupled with enhancer elements from the cytomegalovirus promoter and rabbit β-globin gene (pCAG) and the blasticidin resistance gene driven by the phosphoglycerate kinase (PGK) promoter were generated and electroporated into porcine fetal fibroblasts. Three independent replicates were completed. Following blasticidin selection, the number of surviving colonies was counted to assess the efficiency of the toxic gene. Both NucA and DTA proved to be effective in killing porcine fibroblasts compared to controls. However, the efficiency of cell ablation was significantly higher with DTA than with NucA or NucANLS (p < 0.05). Gene expression analysis of surviving colonies indicated that survival is related to low or absent expression of the toxic genes. These results indicate that the NucA gene, while capable of mammalian cell ablation, is less efficient than DTA.}, number={4}, journal={ANIMAL BIOTECHNOLOGY}, author={Caballero, I. and Piedrahita, J. A.}, year={2009}, pages={177–185} }
 @article{farmer_farin_bischoff_alexander_piedrahita_farin_2008, title={2 DETECTION OF ANTISENSE TO Igf2r (AIR) RNA IN CATTLE}, volume={20}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv20n1Ab2}, DOI={10.1071/RDv20n1Ab2}, abstractNote={ The insulin-like growth factor type 2 receptor (Igf2r) regulates fetal growth by removing Igf2 from circulation, thus preventing overgrowth. In mice, expression of the Igf2r gene is imprinted only after implantation and is associated with expression of the antisense non-coding (nc)RNA, Air. In contrast, the human IGF2R gene is not imprinted and AIR ncRNA does not exist. Because it is known that the Igf2r gene is imprinted in cattle, the objectives of this study were to determine if Air ncRNA exists in cattle and, if so, whether bovine Air (bAir) is expressed during both pre- and post-implantation development. For objective 1, primer sets were designed for bAir based on bovine genomic sequence. The primer set, bAir3, was used to amplify a region of bAir corresponding to an antisense segment within intron 1 of Igf2r. Primer set bAir4 amplified a segment of bAir ncRNA corresponding to an antisense region upstream of the 52-untranslated region of Igf2r. Pools of whole-cell RNA were extracted from bovine fetal liver and subjected to DNase treatment, reverse transcription (RT), and PCR. Control RT reactions included RT without superscript and RT without superscript or DNase. Controls confirmed that amplification products resulted from RNA present in the sample and not from genomic DNA contamination. Amplicons were obtained for both the bAir3 and the bAir4 primer sets and were sequence verified, demonstrating that bAir ncRNA does exist in cattle. For objective 2, conceptuses (n = 4; mean � SEM length: 2.8 � 0.3 mm) derived from transfer of frozen-thawed in vivo-produced blastocysts were recovered from cows on Day 15 of gestation and snap-frozen for RNA extraction. Samples of liver from in vivo-produced bovine fetuses recovered at Day 70 of gestation (n = 7) were snap-frozen for RNA extraction. Semi-quantitative RT-PCR assays were performed to assess levels of mRNA for Igf2r and H2a, as well as ncRNA for bAir. All conceptus and fetal liver cDNA samples were run in duplicate within the same assay. Relative RNA expression was calculated as the ratio of band intensities of the RNA of interest to that of H2a. Data for relative RNA expression were analyzed by Student's t-test. H2a and Igf2r mRNAs were expressed in all Day 70 fetal liver and Day 15 conceptus samples. Relative levels of Igf2r did not differ (P = 0.19) with stage of development (0.15 � 0.09 v. 0.36 � 0.12 for Day 70 v. Day 15). bAir ncRNA was expressed in 7 of 7 samples of Day 70 fetal liver, whereas only 1 of 4 conceptuses expressed a faint bAir ncRNA signal based on either the bAir3 or bAir4 primer sets (χ2 = 7.23, P < 0.01). Relative levels of bAir ncRNA were greater (P < 0.001) in Day 70 fetal liver compared to those in Day 15 conceptuses for amplicons bAir3 (0.376 � 0.039 v. 0.028 � 0.051) and bAir4 (0.101 � 0.008 v. 0.003 � 0.010). In conclusion, the antisense ncRNA, Air, does exist in cattle and its relative expression is greatest following implantation. These observations are consistent with murine data and suggest that bAir may be involved in regulating imprinted expression of Igf2r in cattle.
}, number={1}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Farmer, W. T. and Farin, P. W. and Bischoff, S. R. and Alexander, J. E. and Piedrahita, J. A. and Farin, C. E.}, year={2008}, pages={81} }
 @article{tsai_hardison_bischoff_thames_jamessd_motsinger-reif_piedrahita_2008, title={262: Molecular signatures of preeclampsia in human term placentas}, volume={199}, ISSN={0002-9378}, url={http://dx.doi.org/10.1016/j.ajog.2008.09.290}, DOI={10.1016/j.ajog.2008.09.290}, number={6}, journal={American Journal of Obstetrics and Gynecology}, publisher={Elsevier BV}, author={Tsai, Shengdar and Hardison, Nicholas and Bischoff, Steve and Thames, Betty and Jamessd, Andra and Motsinger-Reif, Alison and Piedrahita, Jorge}, year={2008}, month={Dec}, pages={S84} }
 @article{lee_estrada_piedrahita_2008, title={A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: Effects of the squeezing and the aspiration methods}, volume={19}, ISSN={["1532-2378"]}, DOI={10.1080/10495390701839264}, abstractNote={In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.}, number={2}, journal={ANIMAL BIOTECHNOLOGY}, author={Lee, Eunsong and Estrada, Jose and Piedrahita, Jorge A.}, year={2008}, pages={71–79} }
 @article{zaunbrecher_mir_dunne_breen_piedrahita_2008, title={Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ)}, volume={19}, ISSN={["1532-2378"]}, DOI={10.1080/10495390701670099}, abstractNote={Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.}, number={1}, journal={ANIMAL BIOTECHNOLOGY}, author={Zaunbrecher, Gretchen M. and Mir, Bashir and Dunne, Patrick W. and Breen, Matthew and Piedrahita, Jorge A.}, year={2008}, pages={6–21} }
 @article{bischoff_tsai_hardison_york_freking_nonneman_rohrer_piedrahita_2008, title={Identification of SNPs and INDELS in swine transcribed sequences using short oligonucleotide microarrays}, volume={9}, ISSN={1471-2164}, url={http://dx.doi.org/10.1186/1471-2164-9-252}, DOI={10.1186/1471-2164-9-252}, abstractNote={Abstract}, number={1}, journal={BMC Genomics}, publisher={Springer Nature}, author={Bischoff, Steve R and Tsai, Shengdar and Hardison, Nicholas E and York, Abby M and Freking, Brad A and Nonneman, Dan and Rohrer, Gary and Piedrahita, Jorge A}, year={2008}, pages={252} }
 @article{estrada_collins_york_bischoff_sommer_tsai_petters_piedrahita_2008, title={Successful cloning of the Yucatan minipig using commercial/occidental breeds as oocyte donors and embryo recipients}, volume={10}, ISSN={["1536-2302"]}, DOI={10.1089/clo.2008.0005}, abstractNote={The widespread application of porcine SCNT to biomedical research is being hampered by the large adult size (300-600 lbs) of the commercial breeds commonly used for SCNT. The Yucatan minipig, in contrast, has an adult weight of 140-150 lbs and a long history of utility in biomedical research. In order to combine the wide availability of commercial swine with the biomedical value of the Yucatan minipig, we utilized SCNT using the Yucatan as nuclear donors and commercial swine as both oocyte donors and recipients. Of six recipient gilts receiving 631 SCNT embryos, three went to term and delivered seven piglets, four of which survived to adulthood. Additionally, we obtained fetal fibroblasts from a cloned Yucatan and used them for a second round of SCNT. Of three recipients receiving 315 reconstructed embryos, one went to term and delivered three piglets, one of which survived to adulthood. Both microsatellite and D-loop sequence analysis confirmed that all of the piglets generated were nuclear-mitochondrial hybrids carrying Yucatan nuclear DNA and commercial breed mitochondrial DNA. This report shows that it is possible to produce viable Yucatan SCNT clones and opens up the possibility of developing valuable biomedical models in this porcine breed.}, number={2}, journal={CLONING AND STEM CELLS}, author={Estrada, Jose L. and Collins, Bruce and York, Abby and Bischoff, Steve and Sommer, Jeff and Tsai, Shengdar and Petters, Robert M. and Piedrahita, Jorge A.}, year={2008}, month={Jun}, pages={287–296} }
 @article{estrada_sommer_collins_mir_martin_york_petters_piedrahita_2007, title={Swine generated by somatic cell nuclear transfer have increased incidence of intrauterine growth restriction (IUGR)}, volume={9}, ISSN={["1536-2302"]}, DOI={10.1089/clo.2006.0079}, abstractNote={While somatic cell nuclear transfer (SCNT) has been successful in several species, many pregnancies are lost and anomalies are found in fetal and perinatal stages. In this study SCNT and artificial inseminations (AI) populations were compared for litter size, average birth weight, piglets alive at birth, stillborn, mummies, dead at the first week, intrauterine growth restriction (IUGR) and large for gestational age (LGA). Twenty-three SCNT litters (143 individuals) were compared to 112 AI litters (1300 individuals). Litter size average was 11.5 for AI and 6.2 for SCNT. Litter weight and average birth weight adjusted by litter size were significantly (p < 0.05) higher in AI than in SCNT litters. The SCNT population had a significant (p < 0.01) increase in the number of IUGRs per litter with LSmeans 7.2 +/- 1.4 versus 19.4 +/- 3.5 and means 8.0 +/- 10.8 versus 15.5 +/- 24.5 for AI and SCNT, respectively. Additionally, there was a trend for higher postnatal mortality and stillbirths in the SCNT population. These findings demonstrate that there are some differences between SCNT-derived and AI litters. SCNT-derived pigs are excellent models to study epigenetic factors and genes involved in IUGRs, and to develop effective means to improve fetal growth in humans and animals.}, number={2}, journal={CLONING AND STEM CELLS}, author={Estrada, Jose and Sommer, Jeffrey and Collins, Bruce and Mir, Bashir and Martin, Amy and York, Abby and Petters, Robert M. and Piedrahita, Jorge A.}, year={2007}, pages={229–236} }
 @article{piedrahita_bischoff_estrada_freking_nonneman_martin_mir_rohrer_tsai_2006, title={263 USE OF PORCINE PARTHENOTES AND GENE EXPRESSION PROFILING USING MICROARRAYS FOR IDENTIFICATION OF IMPRINTED GENES}, volume={18}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv18n2Ab263}, DOI={10.1071/RDv18n2Ab263}, abstractNote={Genomic imprinting arises from differential epigenetic markings including DNA methylation and histone modifications and results in one allele being expressed in a parent-of-origin specific manner. For further insight into the porcine epigenome, gene expression profiles of parthenogenetic (PRT; two maternally derived chromosome sets) and biparental embryos (BP; one maternal and one paternal set of chromosomes) were compared using microarrays. Comparison of the expression profiles of the two tissue types permits identification of both maternally and paternally imprinted genes and thus the degree of conservation of imprinted genes between swine and other mammalian species. Diploid porcine parthenogenetic fetuses were generated using follicular oocytes (BOMED, Madison, WI, USA). Oocytes with a visible polar body were activated using a single square pulse of direct current of 50 V/mm for 100 �s and diploidized by culture in 10 �g/mL cycloheximide for 6 h to limit extrusion of the second polar body. Following culture, BP embryos obtained by natural matings, and PRT embryos, were surgically transferred to oviducts on the first day of estrus. Fetuses recovered at 28-30 days of gestation were dissected to separate viscera including brain, liver, and placenta; the visceral tissues were then flash-frozen in liquid nitrogen. Porcine fibroblast tissue was obtained from the remaining carcass by mincing, trypsinization, and plating cells in �-MEM. Total RNA was extracted from frozen tissue or cell culture using RNA Aqueous kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol. Gene expression differences between BP and PRT tissues were determined using the GeneChip� Porcine Genome Array (Affymetrix, Santa Clara, CA) containing 23 256 transcripts from Sus scrofa and representing 42 genes known to be imprinted in human and/or mice. Triplicate arrays were utilized for each tissue type, and for PRT versus BP combination. Significant differential gene expression was identified by a linear mixed model analysis using SAS 5.0 (SAS Institute, Cary, NC, USA). Storey's q-value method was used to correct for multiple testing at q d 0.05. The following genes were classified as imprinted on the basis of their expression profiles: In fibroblasts, ARHI, HTR2A, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, SGCE, SNRPN, and UBE3A; in liver, IGF2, PEG3, PLAGL1, PEG10, and SNRPN; in placenta, HTR2A, IGF2, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, and SNRPN; and in brain, none. Additionally, several genes not known to be imprinted in humans/mice were highly differentially expressed between the two tissue types. Overall, utilizing the PRT models and gene expression profiles, we have identified thirteen genes where imprinting is conserved between swine and humans/mice, and several candidate genes that represent potentially imprinted genes. Presently, our efforts are focused in the identification of single nucleotide polymorphisms (SNPs) to more carefully evaluate the behavior of these genes in normal and abnormal gestations and to test whether the candidate genes are indeed imprinted.
This research was supported by USDA-CSREES grant 524383 to J. P. and B. F.}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Piedrahita, J. and Bischoff, S. and Estrada, J. and Freking, B. and Nonneman, D. and Martin, A. and Mir, B. and Rohrer, G. and Tsai, S.}, year={2006}, pages={239} }
 @article{estrada_lee_piedrahita_2006, title={32 CRYOPRESERVATION OF DONOR CELLS FOR NUCLEAR TRANSFER: EFFECT OF CELL FREEZING METHOD ON THE EFFICIENCY OF SOMATIC CELL NUCLEAR TRANSFER IN PIGS}, volume={18}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv18n2Ab32}, DOI={10.1071/RDv18n2Ab32}, abstractNote={ Donor cell quality is one of the most important factors affecting somatic cell nuclear transfer (SCNT) in mammals. Many studies have been carried out to improve the donor cell characteristics in nuclear transfer, including studies on cell type, cell cycle stage, cell passage, and handling of donor cells before the SCNT. Even though most SCNT work is done with donor cells that have been previously frozen and thawed, no studies have been conducted to evaluate the effect of the cell freezing rate on the SCNT efficiency. The objective of this experiment was to evaluate the effect of the cell freezing method on development of pig SCNT embryos in vitro. Fibroblasts were collected from a 29-day-old female fetus, suspended in DMEM-F12 + 40% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO), and placed in 1.6-mL cryovials for freezing. Vials were randomly assigned to two treatments: In treatment 1, cells were frozen at a controlled rate of 1�C/min in a programmable machine (P) until -40�C, and then plunged into liquid nitrogen (LN2; -196�C). In treatment 2, the traditional system (T), vials were placed in a styrofoam box and left overnight in a freezer at -80�C. The next day samples were plunged into LN2 (196�C). For each treatment, cells were thawed and cultured until confluence before being used for SCNT. Cells were used at passages 2 and 6. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 39 h in TCM 199 supplemented with 10% porcine follicular fluid (pFF), 5 �g/mL insulin, 10 ng/mL epidermal growth factor (EGF), 0.6 mM cysteine, 0.2 mM pyruvate, 25 �g/mL gentamycin and 5 �g/mL each of equine and human chorionic gonadotropin (eCG and hCG). Oocytes were stained with bisbenzimide and enucleated in manipulation media with 7.5 �g/mL cytochalasin B by removing the first polar body and metaphase plate by means of a 16-�m beveled glass pipette. Cells from each treatment were injected into the perivitelline space of recipient enucleated oocytes and fused by two DC pulses of 140 V for 50 �s in fusion media. The fusion rate was evaluated 1 h later, and reconstructed oocytes were activated by two DC pulses of 120 V for 60 �s. After activation, oocytes were placed in bicarbonate-buffered NCSU-13 with 0.4% BSA and cultured at 38.5�C, 5% CO2 in a humidified atmosphere. Embryos were observed for cell cleavage at Day 2, and blastocyst development rate and cell number counting were done at Day 7 of culture. Every experiment was repeated three times. The temperature descending rate for P was slower and more linear (1�C/min vs. 2�C/min) than for the T method. Fusion rate was not significantly affected (P < 0.05) by the freezing method when they were evaluated either individually at each passage or accumulated regardless the passage (78.9 � 3.6% vs. 79.4 � 6.3%) for P and T, respectively. The same trends were observed for cleavage (61.2 � 5.2% vs. 64.3 � 5.2%), blastocyst development (4.2 � 1.8% vs. 5.0 � 2.8%), and number of cells at the blastocyst stage (19.4 � 3.1 vs. 19.8 � 6.2) for P and T, respectively. The present findings indicate that blastocyst development after SCNT does not differ when fetal fibroblasts donor cells are frozen by the two methods tested. }, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Estrada, J. and Lee, E. and Piedrahita, J.}, year={2006}, pages={125} }
 @article{tsai_cassady_freking_nonneman_rohrer_piedrahita_2006, title={Annotation of the Affymetrix(1) porcine genome microarray}, volume={37}, ISSN={["0268-9146"]}, DOI={10.1111/j.1365-2052.2006.01460.x}, abstractNote={Overview: The Affymetrix porcine genome microarray (http:// www.affymetrix.com/products/arrays/specific/porcine.affx) is minimally annotated. Less than 10% of the probe sets on this array are described with gene names, posing a challenge to biological interpretation of data. Lack of annotation is likely due to the limited availability of full-length porcine cDNA sequence. Presented here is a strategy for improving the annotation of this microarray.}, number={4}, journal={ANIMAL GENETICS}, author={Tsai, S. and Cassady, J. P. and Freking, B. A. and Nonneman, D. J. and Rohrer, G. A. and Piedrahita, J. A.}, year={2006}, month={Aug}, pages={423–424} }
 @article{monteiro-riviere_inman_hedgpeth_mosteller_piedrahita_2006, title={Dermatological effects of chronic exposure to 7,12-dimethylbenz[a]anthracene (DMBA) or N-methyl-N-nitrosoguanidine (MNNG) in swine}, volume={25}, ISSN={["1556-9527"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000238451100004&KeyUID=WOS:000238451100004}, DOI={10.1080/15569520600695546}, abstractNote={Purpose: To determine whether chronic exposure to DMBA or MNNG in combination with or without UVB exposure would induce skin carcinomas in swine. Methods: Eight gilts were exposed to 100 mJ of UVB in their left side, allowed to recuperate, and divided into two groups. Each gilt received identical high doses (DMBA 50 µM; MNNG 250 mM), low doses (DMBA 500 nM; MNNG 2.5 mM), carrier (DMSO), or nothing added treatments in the UVB and non-UVB sides. Animals were exposed weekly for 30 weeks and skin samples collected at 10, 20, and 30 weeks from initiation of exposure. An additional sample was collected 16 weeks following cessation of exposure. All samples were scored for dermal morphology, including intracellular epidermal edema, intercellular epidermal edema, papillary dermal edema, perivascular infiltrates, pyknotic stratum basale cells, collagen necrosis, and epidermal-dermal separation, and the data were analyzed by ANOVA. MNNG and UVB light had a significant effect on epidermal thickness and the number of cell layers. The greatest increase in epidermal thickness occurred from 20 weeks to 30 weeks in the UVB plus MNNG treatment. Treatment with MNNG resulted in intracellular and intercellular epidermal edema, dermal edema, and dermal inflammation at both the low and high doses of MNNG. In contrast, all the morphological evaluations of the DMBA treatments were less severe than the MNNG. Conclusion: Our findings show that although chronic exposure to MNNG and DMBA, with or without UVB exposure, caused severe to mild dermatopathological changes, neither resulted in the development of skin carcinomas. These results indicate that at least with respect to responses to DMBA and MNNG, the swine model mimics more closely the responses seen in human skin.}, number={2}, journal={CUTANEOUS AND OCULAR TOXICOLOGY}, author={Monteiro-Riviere, N and Inman, A and Hedgpeth, V and Mosteller, B and Piedrahita, J}, year={2006}, pages={103–119} }
 @article{tsai_mir_martin_estrada_bischoff_hsieh_cassady_freking_nonneman_rohrer_et al._2006, title={Detection of transcriptional difference of porcine imprinted genes using different microarray platforms}, volume={7}, journal={BMC Genomics}, author={Tsai, S. and Mir, B. and Martin, A.C. and Estrada, J.L. and Bischoff, S.R. and Hsieh, W.P. and Cassady, J.P. and Freking, B.A. and Nonneman, D.J. and Rohrer, G.A. and et al.}, year={2006}, pages={328–336} }
 @article{farin_piedrahita_farin_2006, title={Errors in development of fetuses and placentas from in vitro-produced bovine embryos}, volume={65}, ISSN={["1879-3231"]}, DOI={10.1016/j.theriogenology.2005.09.022}, abstractNote={In vitro systems for oocyte maturation, fertilization and embryo culture [in vitro production (IVP)] have the potential for more wide-spread use in creative breeding programs for dairy and beef cattle. However, one negative consequence of both IVP and somatic cell nuclear transfer (SCNT) in cattle and other species is that embryos, fetuses, placentas, and offspring can differ significantly in morphology and developmental competence compared with those from embryos produced in vivo. Fetuses and placentas derived from IVP and SCNT embryos may fall within the normal range of development, may have obvious abnormalities such as increased fetal and placental weights, or may have subtle abnormalities such as aberrant development of fetal skeletal muscle, placental blood vessels, and altered metabolism. Failures in physiologic and/or genetic mechanisms essential for proper fetal growth and survival outside of the uterus contribute significantly to pregnancy and neonatal losses. Oversized fetuses are at increased risk of death during parturition and the adverse consequences of severe dystocia may compromise the dam. Collectively, these abnormalities have been referred to as ‘large offspring syndrome’ or ‘large calf syndrome’. Abnormal phenotypes resulting from IVP and SCNT embryos are stochastic in occurrence and they have not been consistently linked to aberrant expression of single genes or specific pathophysiology. Thus, reliable methods of early diagnosis of the condition are not yet available. The objective of this paper is to examine abnormal development of fetuses and placentas resulting from embryos produced using in vitro systems. The term ‘abnormal offspring syndrome (AOS)’ is introduced and a classification system of developmental outcomes is proposed to facilitate research efforts on the mechanisms of the various abnormal phenotypes. We also discuss potential genetic and physiologic mechanisms that may contribute to abnormal phenotypes following transfer of IVP and SCNT embryos.}, number={1}, journal={THERIOGENOLOGY}, author={Farin, PW and Piedrahita, JA and Farin, CE}, year={2006}, month={Jan}, pages={178–191} }
 @article{mir_zaunbrecher_piedrahita_2005, title={51 PHENOTYPIC VARIATION IN CLONED SWINE IS CORRECTED IN THE F1 GENERATION}, volume={17}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv17n2Ab51}, DOI={10.1071/RDv17n2Ab51}, abstractNote={
Systematic studies of cloned animals generated from adult somatic cell nuclei are critical in assessing the utility of somatic cell cloning in various applications, including the safety of food products from cloned animals and their offspring. Studies in mice show that abnormalities seen in the cloned parents are not transmitted to the siblings. To our knowledge, however, there are no studies on the F1 progeny of clones from food animals. Previously, we compared somatic cell derived cloned pigs with naturally bred control pigs on a series of physiological and genetic parameters. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. We have extended our studies to the F1 progeny of these clones to see whether these phenotypic differences are transmitted to the next generation. Age-, sex-, and breed-matched cloned and control pigs, housed together since weaning, were used in this study. Starting with their second estrus cycle, standing gilts were mated two consecutive days. All gilts were mated to the same boar. Pregnant cloned (n = 9) and naturally bred (n = 5) gilts (F0) were allowed to farrow naturally, and number and sex of live offspring at birth (F1) recorded. There was no difference in the average litter size between litters from cloned gilts and naturally bred controls (7.78 ± 2.6 and 7.40 ± 3.0, respectively; mean ± SD) or in the degree of litter size variation (coefficients of variation of 33.4% and 40.5% for litters of clones and controls, respectively). Similarly there were no statistical differences between sex ratios from cloned litters (51%:49%; M:F) and control litters (59%:41%; M:F). Blood profiles among cloned pigs, control pigs, and their progeny were compared at two time points, i.e. 15 and 27 weeks, to quantify the effect of cloning on various blood parameters and their transmission to next generation. Although the range of values for all traits overlapped between different classes, the variation differed between F0 clones and F0 controls. In the clones there were two groups of traits: one in which cloned pigs had less variability than controls, and the other in which clones had the same variability as control pigs. In contrast, the variability between all of the traits in F1 progeny of both the clones and the control pigs was similar at 15 and 27 weeks, with one exception. Combined, our data and previous results in mice strongly support the hypothesis that offspring of clones are to all intent and purposes indistinguishable from offspring of naturally bred animals, and as such there should not be any increased risks associated with consumption of products from these animals.
This work was supported from NIH grant HL 51587 to JAP.}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Mir, B. and Zaunbrecher, G. and Piedrahita, J.A.}, year={2005}, pages={175} }
 @article{spiegelstein_gould_wlodarczyk_tsie_lu_le_troen_selhub_piedrahita_salbaum_et al._2005, title={Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies}, volume={203}, ISSN={["1096-0333"]}, DOI={10.1016/j.taap.2004.07.006}, abstractNote={Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2-/-) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2-/- and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.}, number={1}, journal={TOXICOLOGY AND APPLIED PHARMACOLOGY}, author={Spiegelstein, O and Gould, A and Wlodarczyk, B and Tsie, M and Lu, XF and Le, C and Troen, A and Selhub, J and Piedrahita, JA and Salbaum, JM and et al.}, year={2005}, month={Feb}, pages={18–26} }
 @article{ma_finnell_davidson_callaway_spiegelstein_piedrahita_salbaum_kappen_weeks_james_et al._2005, title={Folate transport gene inactivation in mice increases sensitivity to colon carcinogenesis}, volume={65}, number={3}, journal={Cancer Research}, author={Ma, D. W. L. and Finnell, R. H. and Davidson, L. A. and Callaway, E. S. and Spiegelstein, O. and Piedrahita, J. A. and Salbaum, J. M. and Kappen, C. and Weeks, B. R. and James, J. and et al.}, year={2005}, pages={887–897} }
 @article{mir_zaunbrecher_archer_friend_piedrahita_2005, title={Progeny of somatic cell nuclear transfer (SCNT) pig clones are phenotypically similar to non-cloned pigs}, volume={7}, ISSN={["1536-2302"]}, DOI={10.1089/clo.2005.7.119}, abstractNote={Systematic studies of cloned animals generated from adult somatic cell nuclei are critical in assessing the utility of somatic cell cloning in various applications, including the safety of food products from cloned animals and their offspring. Previously, we compared somatic cell derived cloned pigs with naturally bred control pigs on a series of physiological and genetic parameters. We have extended our studies to the F1 progeny of these clones to see whether these phenotypic differences are transmitted to the next generation. There were no differences in the average litter size between litters from cloned gilts and naturally bred controls (7.78 +/- 2.6 and 7.40 +/- 3.0, respectively; mean +/- SD) or in the degree of litter size variation (coefficients of variation of 33.4% and 40.5% for litters of clones and controls, respectively). Similarly there were no statistical differences between sex ratios of cloned litters (51-49%, M:F) and control litters (59-41%, M:F). Blood profiles between cloned pigs, control pigs, and their progeny were compared at two time points (i.e., 15 and 27 weeks) to quantify the effect of cloning on various blood parameters and their transmission to the next generation. Although the range of values for all traits overlapped between different classes, the variability between all the traits in F1 progeny of clones and the control pigs was similar at 15 and 27 weeks, with one exception. Combined, our data and previous results in mice strongly support the hypothesis that offspring of clones are similar to offspring of naturally bred animals, and as such there should not be any increased risks associated with consumption of products from these animals.}, number={2}, journal={CLONING AND STEM CELLS}, author={Mir, B and Zaunbrecher, G and Archer, GS and Friend, TH and Piedrahita, JA}, year={2005}, pages={119–125} }
 @article{mir_piedrahita_2004, title={343NUCLEAR LOCALIZATION SIGNAL AND CELL SYNCHRONY ENHANCES GENE TARGETING 
EFFICIENCY IN FETAL BOVINE FIBROBLASTS}, volume={16}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv16n1Ab343}, DOI={10.1071/RDv16n1Ab343}, abstractNote={
The use of primary somatic cells for nuclear transfer has facilitated the manipulation of the domestic animal genome via homologous recombination. Yet, the absolute frequency of homologous recombination (HR) in somatic cells is two orders of magnitude lower than in ES cells whereas frequencies of non-homologous end joining are higher. While a few loci have been targeted in somatic cells using enrichment strategies similar to those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells, and difficulties in cloning the cell after targeting. We present evidence that the use of a nuclear localization signal (nls) and S-phase cell cycle synchronization by thymidine block enhances targeting efficiency at the hypoxanthine phosphoribosyl transferase locus in primary fetal bovine fibroblasts. We designed two hypoxanthine phosphoribosyl transferase (HPRT)-targeting constructs, HPRT-DEx6 and HPRT-DEx6-nls. Both constructs have a 31-bp deletion and a PGK-puro insertion in exon 6 to ensure inactivation of the HPRT locus. Additionally, the HPRT-DEx6-nls construct contains a 180-bp cassette comprised of two 72-bp tandem repeats from SV40 enhancer known to act as nuclear localization signal. Diploid male cells that undergo targeted gene disruption at the single copy X-linked HPRT locus can be selected with 8-Azaguanine (8-AG) as HPRT cells are resistant to 8-AG; all transformants, random and targeted, can be selected in puromycin. Male primary bovine fibroblasts were electroporated with linearized targeting constructs, and plated in media containing puromycin or puromycin plus 8AG. All experiments were done in triplicate and data were analyzed by two-way ANOVA with NLS and cell synchrony as the two factors. Significance was set at P<0.01. While the total number of insertions (random plus targeted) with both constructs were equivalent, the HPRT-DEx6-nls construct produced a significantly higher number of targeted colonies (1–2 8AG-resistant colonies per 9.5×106 cells) than HPRT-DEx6 where no targeted events were seen. Cells were synchronized in the S-phase of the cell cycle by a 2mM thymidine treatment for 24 hours and electroporated with the linearized targeting constructs. Compared to non-synchronous cells, the total number of insertions (random plus targeted) was reduced by 59-fold in constructs with or without nls (P<0.01), while targeted insertions increased 6-fold in the HPRT-DEx6-nls construct, from an average of 10 per million cells without nls to 7.6 per 10 million with nls (P<0.01). All 8AG-resistant colonies were verified by Long Range-PCR, and PCR products confirmed by end sequencing. This finding has important implications for targeting in somatic cells, as with a drastic reduction in the number of random insertions, and increased targeting due to the presence of the nls, identification of a targeted colony is greatly facilitated even in cases where no enrichment protocols are available.
}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Mir, B. and Piedrahita, J.A.}, year={2004}, pages={291} }
 @article{dindot_farin_farin_alexander_crosier_walker_long_piedrahita_2004, title={35 Analysis of epigenetic modifications and genomic imprinting in nuclear transfer derived Bos Gaurus x B. Taurus Concepti}, volume={16}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv16n1Ab35}, DOI={10.1071/RDv16n1Ab35}, abstractNote={
Somatic cell nuclear transfer in cattle is an inefficient process hindered by low pregnancy rates and fetal placental abnormalities. Improper or incomplete epigenetic reprogramming of the donor genome has been implicated as a cause for these aberrations and has been investigated extensively in mice. Here we report the use of a bovine interspecies model (Bos gaurus×B. taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in 40-day-old female nuclear transfer (NT)-derived fetuses and placentas. Previously, we identified genomic imprinting at the IGF2, GTL2 and XIST loci in the Bos gaurus×B. taurus fetuses. These results indicated maternal and paternal imprinting of the IGF2 and GTL2 loci, respectively, in the chorion, allantois, liver, lung and brain, whereas the XIST locus was maternally imprinted solely in the chorion of females. We extended this analysis to 40-day-old NT fetuses derived from a hybrid lung fibroblast cell line (female). Analysis of the donor cell line indicated conservation of imprinting of the IGF2 and GTL2 loci and bialleic expression of the XIST locus, presumably from the random patterns of X-chromosome inactivation. Analysis of three NT and three control pregnancies indicated disruption of genomic imprinting at the XIST locus in the chorions of all three clones compared to controls. In contrast, proper allelic expression of the IGF2 and GTL2 loci was observed in all fetuses and placentas. Quantification of maternal and paternal XIST transcripts in the chorion of clones and controls demonstrated a significant skewing from preferential paternal expression in controls (95.0±0.882, mean±S.E.) to mixed paternal and maternal expression in clones (73.6±5.2), (t-test; P<0.05). In an attempt to determine the cause for the abnormal allelic expression of the XIST locus in the chorion of the clones, methylation analysis of the XIST Differentially Methylated Region (DMR) was performed. Methylation-sensitive restriction digests and subsequent PCR of the XIST DMR indicated patterns were not different between controls and clones. However, when genome-wide and promoter-specific methylation analysis (bisulfite sequencing) was extended to the satellite I repeat element and epidermal cytokeratin promoter, hypermethylation was observed in the chorion of clones. These results demonstrate disruption of genomic imprinting in XIST locus in the placenta of 40-day-old clones independent of DMR methylation. They also indicate that cloning is associated with increased levels of methylation in selected genomic regions in the chorion.
}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Dindot, S.V. and Farin, P. and Farin, C. and Alexander, J. and Crosier, E. and Walker, S. and Long, C. and Piedrahita, J.A.}, year={2004}, pages={140} }
 @article{piedrahita_walker_zaunbrecher_2004, title={67ORGAN WEIGHT VARIATION IN CLONED PIGS}, volume={16}, ISSN={1031-3613}, url={http://dx.doi.org/10.1071/RDv16n1Ab67}, DOI={10.1071/RDv16n1Ab67}, abstractNote={
Previously, we compared adult clones and naturally bred control females using a series of physiological and genetic parameters and found considerable variation in certain traits in clones. In order to more fully understand this clonal variation we examined the organ weight differences among a group of clones from a single litter. Briefly, in vitro-matured oocytes were stripped of their cumulus cells at 46–48h postmaturation by vortexing in TCM 199 containing 5% FCS with 0.1% hyaluronidase, and placed in Ca-free NCSU-23 with 4mgmL−1 BSA. Oocytes were stained in Ca-free NCSU-23 containing 4mgmL−1 BSA and 5μgmL−1 Hoechst 3334 for 10min, and enucleated in Ca-free, salt buffered, NCSU-23 containing 5% FCS and 7.5μgmL−1 of cytochalasin B, Puromycin-resistant fetal fibroblasts grown for 35 days in D-MEM/F-12 containing 15% FCS and 1.65μgmL−1 puromycin were synchromized by contact inhibition for 1-2 days. Cells were trypsinized, resuspended in salt buffered NCSU-23 with 10% FCS and placed into the perivitelline space. Couplets wer fused by 2V AC for 2s followed by 2 pulses DC of 1.4kvcm−1 for 50μs. Following fusion, the oocytes were placed in NCSU-23 containing 4mgmL−1 BSA for 1.5–2h. The oocytes were then electroactivated by 2 pulses of 1.2kgcm−1 for 50μs, allowed to recuperate for 10–15min, and transferred into the oviduct of a naturally synchronized recipient. A total of 286 doublets were transferred into 4 recipients one of which became pregnant and gave birth to 9 clones (9/81). One clone died soon after birth. The remaining 8 clones were weaned at three weeks and kept as a group until Day 120 at which time the animals were euthanized for organ recovery. Weights were determined for major organs, and coefficients of variation (CV) calculated for both absolute weights, and corrected total weight. Corrected weights were calculated as weight of organ divided by weight of clone. CV for absolute and corrected weights, respectively, were: uterus, 15.1% and 5%; left kidney, 13.2% and 6.9%; right kidney, 11% and 6.8%; spleen, 29.7% and 27.1%; liver, 20.6% and 19.7%; lung, 37.5% and 32%; heart, 20.2% and 15.5%; and brain, 5.6% and 9.5%. Both absolute and corrected weights indicated that there is a high degree of variation in some of the organs among a group of identical clones. To determine whether the variation we observed is typical of non-cloned swine, we compared the variations in the clones to those observed in a group of 3 age-matched, sex-matched, breed-matched controls that had been kept with the clones for approximately 90 days. Relative coefficients of variation of corrected organ weights between clones and non-cloned animals were: uterus, 0.18; left kidney, 0.34; right kidney, 0.34; spleen, 2.58; liver, 1.08; lung, 1.06; heart, 0.59; and brain, 0.7. In summary, these results indicate that some organs in cloned animals have a high degree of weight variability, most likely due to epigenetic aberrations of overriding environmental effects.
}, number={2}, journal={Reproduction, Fertility and Development}, publisher={CSIRO Publishing}, author={Piedrahita, J.A. and Walker, S. and Zaunbrecher, G.}, year={2004}, pages={155} }
 @article{piedrahita_mir_2004, title={Cloning and transgenesis in mammals: Implications for xenotransplantation}, volume={4}, ISSN={["1600-6143"]}, DOI={10.1111/j.1600-6135.2004.0344.x}, abstractNote={Availability of suitable organs for transplantation remains of major concern and projections indicate that the problem will continue to increase. Therefore, alternatives to the use of human organs for transplantation, continue to be explored including use of stem cells, artificial organs, and organs from other species (xenotransplantation). In xenotransplantation, the species of choice remains the pig due to its physiological similarities to humans, reduced costs, ease of manipulation, and reduced ethical concerns to its use. However, in order to develop pig organs that are suitable for xenotransplantation, complex genetic modification need to be undertaken. These modifications require the introduction of precise genetic changes into the pig that can only be accomplished at this time using somatic cell nuclear transfer. We cover in this review advances in transgenic manipulation and cloning in swine and how the development of these two technologies is critical to the eventual utilization of the pig as a human organ donor.}, journal={AMERICAN JOURNAL OF TRANSPLANTATION}, author={Piedrahita, JA and Mir, B}, year={2004}, month={Feb}, pages={43–50} }
 @article{seabury_womack_piedrahita_derr_2004, title={Comparative PRNP genotyping of US cattle sires for potential association with BSE}, volume={15}, ISSN={["1432-1777"]}, DOI={10.1007/s00335-004-2400-6}, abstractNote={The recent discovery of significant associations between bovine spongiform encephalopathy (BSE) susceptibility in German cattle and the frequency distributions of insertion/deletion (indel) polymorphisms within the bovine PRNP gene prompted an evaluation of 132 commercial U.S. artificial insemination (AI) sires from 39 breeds. Forward primer sequences from published primer sets targeting indels within the putative bovine PRNP promoter, intron 1, and the 3' UTR (untranslated region) were synthesized with unique 5' fluorescent labels and utilized to develop a rapid multiplexed PCR assay for identifying BSE-associated indels as well as facilitating polymorphism analyses and/or marker-assisted selection. Significant differences ( p < 0.05 all tests) were detected between the frequencies of bovine PRNP promoter alleles for 48 healthy German cattle previously described and 132 commercial U.S. cattle sires. The frequency of the 23-bp promoter allele observed for commercial U.S. cattle sires strongly resembled that recently described for 43 BSE-affected German cattle. No significant difference ( p = 0.051) was detected between the distributions of promoter genotypes for healthy German cattle and our panel of commercial U.S. cattle sires. Interestingly, significant differences ( p < 0.01; p < 0.02) were also noted between the frequencies and distributions of intron 1 alleles and genotypes, respectively, for BSE-affected German cattle and our panel of U.S. cattle sires. No significant allelic or genotypic differences were detected for the 14-bp 3' UTR indel for any given comparison between German cattle and commercial U.S. cattle sires.}, number={10}, journal={MAMMALIAN GENOME}, author={Seabury, CM and Womack, JE and Piedrahita, J and Derr, JN}, year={2004}, month={Oct}, pages={828–833} }
 @article{dindot_kent_evers_loskutoff_womack_piedrahita_2004, title={Conservation of genomic imprinting at the XIST, IGF2, and GTL2 loci in the bovine}, volume={15}, ISSN={["1432-1777"]}, DOI={10.1007/s00335-004-2407-z}, abstractNote={Genomic imprinting is theorized to exist in all placental mammals and some marsupials; however, extensive comparative analysis of animals aside from humans and mice remains incomplete. Here we report conservation of genomic imprinting in the bovine at the X chromosome inactivation-specific transcript (XIST), insulin-like growth factor 2 (IGF2), and gene trap locus 2 (GTL2) loci. Coding single nucleotide polymorphisms (SNPs) between Bos gaurus and Bos taurus were detected at the XIST, IGF2, and GTL2 loci, which have previously been identified as imprinted in either humans, mice, or sheep. Expression patterns of parental alleles in F1 hybrids indicated preferential paternal expression at the XIST locus solely in the chorion of females, whereas analysis of the IGF2 and GTL2 loci indicated preferential paternal and maternal expression of alleles, respectively, in both fetal and placental tissues. Comparative sequence analysis of the XIST locus and adjacent regions suggests that repression of the maternal allele in the bovine is controlled by a different mechanism than in mice, further reinforcing the importance of comparative analysis of imprinting.}, number={12}, journal={Mammalian Genome}, author={Dindot, S. and Kent, K.C. and Evers, B. and Loskutoff, N. and Womack, J. and Piedrahita, J.A.}, year={2004}, pages={966–974} }
 @article{farin_farin_piedrahita_2004, title={Development of fetuses from in vitro produced and cloned bovine embryos}, volume={82}, DOI={10.2527/2004.8213_supplE53x}, abstractNote={The establishment of in vitro fertilization and culture systems for mammalian embryos has facilitated the application of embryo technologies in research, industry, and clinical settings. Furthermore, the advent of cloning by nuclear transfer has significantly enhanced the potential for genetic modification of livestock. Based on studies in cattle, sheep, and mice, it has become apparent that embryos produced using these systems can differ in morphology and developmental potential compared with embryos produced in vivo. Referred to as "large offspring syndrome," these abnormalities in the development of fetuses, placentas, and offspring are particularly evident following transfer of cloned embryos, but they also occur in pregnancies from embryos produced using in vitro culture alone. The objective of this review is to examine the effects of in vitro production and cloning on bovine embryo and fetal development. Literature pertaining to preimplantation embryo, conceptus, and fetal development, as well as gene expression occurring at each of these three stages, is reviewed. Physiologic and genetic mechanisms that contribute to large offspring syndrome also are discussed.}, number={Supplement 13}, journal={Journal of Animal Science}, author={Farin, C. and Farin, P.W. and Piedrahita, J.A.}, year={2004}, month={Jan}, pages={E53–E62} }
 @article{dindot_farin_farin_romano_walker_long_piedrahita_2004, title={Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses}, volume={71}, DOI={10.1095/biolreprod.103.025775}, abstractNote={Abstract Somatic cell nuclear transfer (NT) in cattle is an inefficient process, whereby the production of calves is hindered by low pregnancy rates as well as fetal and placental abnormalities. Interspecies models have been previously used to facilitate the identification of single nucleotide polymorphisms (SNPs) within coding regions of genes to discriminate between parental alleles in the offspring. Here we report the use of a bovine interspecies model (Bos gaurus × Bos taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in Day 40-old female NT-derived fetuses and placenta. Analysis of NT and control pregnancies indicated disruption of genomic imprinting at the X inactivation-specific transcript (XIST) locus in the chorion, but not the fetus of clones, whereas proper allelic expression of the insulin-like growth factor II (IGF2) and gene trap locus 2 (GTL2) loci was maintained in both the fetus and placenta. Analysis of the XIST differentially methylated region (DMR) in clones indicated normal patterns of methylation; however, bisulfite sequencing of the satellite I repeat element and epidermal cytokeratin promoter indicated hypermethylation in the chorion of clones when compared with controls. No differences were detected in methylation levels in the fetus proper. These results indicate that the nuclear transfer process affects gene expression patterns in the trophectoderm- and inner cell mass-derived tissues to different extents.}, number={2}, journal={Biology of Reproduction}, author={Dindot, S. V. and Farin, P. W. and Farin, C. E. and Romano, J. and Walker, S. and Long, C. and Piedrahita, Jorge}, year={2004}, pages={470–478} }
 @article{mir_piedrahita_2004, title={Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts}, volume={32}, DOI={10.1093/nar/gnh023}, abstractNote={The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date, while a few loci have been targeted in somatic cells using similar enrichment strategies as those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells within a colony and difficulties in cloning the cell after targeting. Utilizing the hypoxanthine guanine phosphoribosyl transferase (HPRT) as a test locus, it was determined that while no targeted colonies were identified using a conventional targeting construct, an average of 1 per million targeted cells were identified when a nuclear localization signal (nls) was added to the construct. When the nls was combined with cell synchronization using a thymidine block, targeting efficiency increased 7-fold. Moreover, the number of random integrants decreased by over 54-fold resulting in a 1:3 targeted to random integration ratio. This method should facilitate the application of homologous recombination to primary somatic cells.}, number={3}, journal={Nucleic Acids Research}, author={Mir, B. and Piedrahita, J.A.}, year={2004}, month={Feb}, pages={e25–e28} }
 @article{piedrahita_mir_dindot_walker_2004, title={Somatic cell cloning: The ultimate form of nuclear reprogramming?}, volume={15}, ISSN={["1046-6673"]}, DOI={10.1097/01.ASN.0000110183.87476.05}, abstractNote={With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.}, number={5}, journal={JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY}, author={Piedrahita, JA and Mir, B and Dindot, S and Walker, S}, year={2004}, month={May}, pages={1140–1144} }
 @article{benjamin_jenster_piedrahita_2004, title={Use of artificial androgen receptor coactivators to alter myoblast proliferation}, volume={91}, ISSN={["0960-0760"]}, DOI={10.1016/j.jsbmb.2004.02.007}, abstractNote={Skeletal muscle has long been thought to be a target tissue for androgens, eliciting their effect through the androgen receptor. In order to better understand androgen receptor action, a series of mutated androgen receptors were developed and their degree of specificity and cellular responses determined. Specificity, as measured by a reporter assay using HeLa cells, indicated that mutation of the ligand-binding domain or the AR (mutation H865Y), in combination with the p65 transactivating domain, resulted in an increased response to androgens as well as decreased specificity. Transfection of the mutant AR into mouse and rat myoblast cell lines resulted in an increase in expression of the reporter gene consistent with the data from HeLa cells. Overexpression of the wild type or mutant AR into myoblasts and treatment with testosterone induced both greater proliferation and faster differentiation of the cells compared to those expressing endogenous AR. Additionally, when treated with estrogen, these cells were able to proliferate and differentiate to similar levels as cells treated with testosterone. The ability of the mutated AR to act as an artificial coactivator to up-regulate androgen responsive genes is a useful tool for understanding the interaction of androgens and muscle growth.}, number={3}, journal={JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY}, author={Benjamin, CL and Jenster, G and Piedrahita, JA}, year={2004}, month={Jul}, pages={111–119} }
 @article{archer_friend_piedrahita_nevill_walker_2003, title={Behavioral variation among cloned pigs}, volume={81}, ISSN={["0168-1591"]}, DOI={10.1016/S0168-1591(02)00272-1}, abstractNote={The variability of behavior among cloned animals has yet to be studied. Through a series of behavior tests, we quantified the variation in food preference, temperament, and time budgets of two genetically identical Duroc litters (n=5, 4) and their naturally bred controls (n=4, 4). All litters of pigs were tested for their food preference using apples, bananas, crackers, and carrots. Variation in temperament was determined by timing latency to remove a towel (Towel Test) and by counting vocalizations and escape attempts during Back and Pick-up Tests. Seventy-two hours of time lapse video were used to determine time budgets of the pigs consisting of the following behaviors: lying in bedding, lying on concrete, standing, feeding, and play/fighting. An F-test was used to determine differences in variation between litter variations. The clones were similarly or more variable (P<0.05) than the naturally bred controls: in their preference for the foods in 13 of the 160 comparisons; in 5 of the 8 comparisons during the Towel Test; in all four comparisons in the Back and Pick-up Tests; and in 9 of the 10 comparisons in the time budget analysis. These results reinforce the importance of environmental effects on animal behavior and question the use of cloning by nuclear transfer to replicate animals with specific behavioral characteristics.}, number={4}, journal={Applied Animal Behaviour Science}, author={Archer, G. and Friend, T. and Piedrahita, J.A. and Nevill, C. and Walker, S.C.}, year={2003}, month={May}, pages={321–331} }
 @article{archer_friend_piedrahita_nevill_walker_2003, title={Behavioral variation among cloned pigs}, volume={82}, DOI={10.1016/S0168-1591(03)00065-0}, abstractNote={The variability of behavior among cloned animals has yet to be studied. Through a series of behavior tests, we quantified the variation in food preference, temperament, and time budgets of two genetically identical Duroc litters (n=5, 4) and their naturally bred controls (n=4, 4). All litters of pigs were tested for their food preference using apples, bananas, crackers, and carrots. Variation in temperament was determined by timing latency to remove a towel (Towel test) and by counting vocalizations and escape attempts during Back and Pick-up tests. Seventy-two hours of time lapse video were used to determine time budgets of the pigs consisting of the following behaviors: lying in bedding, lying on concrete, standing, feeding, and play/fighting. An F-test was used to determine differences in variation between litter variations. The clones were similarly or more variable (P<0.05) than the naturally bred controls: in their preference for the foods in 13 of the 16 comparisons; in 5 of the 8 comparisons during the Towel test; in all four comparisons in the Back and Pick-up tests; and in 9 of the 10 comparisons in the time budget analysis. These results reinforce the importance of environmental effects on animal behavior and question the use of cloning by nuclear transfer to replicate animals with specific behavioral characteristics.}, number={2}, journal={Applied Animal Behaviour Science}, author={Archer, G. and Friend, T. and Piedrahita, J.A. and Nevill, C. and Walker, S.C.}, year={2003}, month={Jun}, pages={151–161} }
 @article{archer_dindot_friend_walker_zaunbrecher_lawhorn_piedrahita_2003, title={Hierarchical phenotypic and epigenetic variation in cloned swine}, volume={69}, ISSN={["0006-3363"]}, DOI={10.1095/biolreprod.103.016147}, abstractNote={Abstract Cloning by somatic cell nuclear transfer can result in the birth of animals with phenotypic and gene expression abnormalities. We compared adult cloned pigs and adult pigs from naturally bred control females using a series of physiological and genetic parameters, including detailed methylation profiles of selected genomic regions. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. Although cloning creates animals within the normal phenotypic range, it increases the variability associated with some traits. This finding is contrary to the expectation that cloning can be used to reduce the size of groups involved in animal experimentation and to reproduce an animal, including a pet, with a homogenous set of desired traits.}, number={2}, journal={BIOLOGY OF REPRODUCTION}, author={Archer, GS and Dindot, S and Friend, TH and Walker, S and Zaunbrecher, G and Lawhorn, B and Piedrahita, JA}, year={2003}, month={Aug}, pages={430–436} }
 @article{kim_song_piedrahita_2003, title={Kidney-specific activity of the bovine uromodulin promoter}, volume={12}, ISSN={["0962-8819"]}, DOI={10.1023/A:1022911124946}, abstractNote={A 10-kilobase (kb) lambda bacteriophage bovine genomic clone containing 5.4 kb of the 5'-flanking region, exons, and introns of bovine uromodulin gene was isolated. Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection. RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney. In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney. Stepwise 5' deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5'-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region.}, number={2}, journal={TRANSGENIC RESEARCH}, author={Kim, HT and Song, IY and Piedrahita, J}, year={2003}, month={Apr}, pages={191–201} }
 @article{thomson_rucker_piedrahita_2003, title={Mutational analysis of LoxP sites for efficient Cre-mediated insertion into genomic DNA}, volume={36}, ISSN={["1526-954X"]}, DOI={10.1002/gene.10211}, abstractNote={Abstract}, number={3}, journal={GENESIS}, author={Thomson, JG and Rucker, EB and Piedrahita, JA}, year={2003}, month={Jul}, pages={162–167} }
 @article{mir_tanner_chowdhary_piedrahita_2003, title={UP1 extends life of primary porcine fetal fibroblasts in culture}, volume={5}, ISSN={["1536-2302"]}, DOI={10.1089/153623003322234740}, abstractNote={Genetic modification of somatic cell nuclei and subsequent nuclear transfer has opened an opportunity to create gene-targeted animals. However, somatic cells have a limited life span in culture and it is not possible to introduce precise genetic changes in both alleles in this narrow time window. To increase the life span of somatic cell in culture, both genetic and chemical approaches have been tried with varying success. Here, we report the effect of two anti-oxidants, glutathione and n-t-butyl hydroxylamine, and of the expression of UP1, a shortened derivative of heterogeneous nuclear riboprotein (hnRNP)A1, on the life extension of primary porcine fibroblasts in culture. Under our experimental conditions, the use of anti-oxidants did not result in any prolongation of the life span. In contrast, UP1 expression increased the life span significantly. While most control cells stopped growing by PDL 20, and none survived beyond PDL 35, 100% of UP1-expressing clones reached PDL50, and 40% made it to PDL65. The five UP1-expressing clones were karyotyped at PDL 50. While all of them had a range of numerical chromosomal abnormalities, two clones retained 30-40% normal cells, all the cells in other three clones had abnormal chromosome numbers. Thus, expression of UP1 may be useful in extending the life span of somatic cells in culture. This, in turn, will facilitate the process of gene targeting in this cell type.}, number={2}, journal={CLONING AND STEM CELLS}, author={Mir, B and Tanner, N and Chowdhary, BP and Piedrahita, JA}, year={2003}, pages={143–148} }
 @article{walker_shin_zaunbrecher_romano_johnson_bazer_piedrahita_2002, title={A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes}, volume={2}, DOI={10.1089/153623002320253283}, abstractNote={To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.}, journal={Cloning and Stem Cells}, author={Walker, S. C. and Shin, T. Y. and Zaunbrecher, G. M. and Romano, J. E. and Johnson, G. A. and Bazer, F. W. and Piedrahita, Jorge}, year={2002}, pages={105–112} }
 @article{piedrahita_wells_miller_oliver_berg_peterson_tervit_2002, title={Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle}, volume={61}, ISSN={["1098-2795"]}, DOI={10.1002/mrd.10013}, abstractNote={Abstract}, number={3}, journal={MOLECULAR REPRODUCTION AND DEVELOPMENT}, author={Piedrahita, JA and Wells, DN and Miller, AL and Oliver, JE and Berg, MC and Peterson, AJ and Tervit, HR}, year={2002}, month={Mar}, pages={317–326} }
 @article{lee_piedrahita_2002, title={Inhibition of apoptosis in serum starved porcine embryonic fibroblasts}, volume={62}, ISSN={["1098-2795"]}, DOI={10.1002/mrd.10058}, abstractNote={Abstract}, number={1}, journal={MOLECULAR REPRODUCTION AND DEVELOPMENT}, author={Lee, CK and Piedrahita, JA}, year={2002}, month={May}, pages={106–112} }
 @article{lee_piedrahita_2001, title={Inactivation of the whey acidic protein (WAP) gene by site-specific recombination in mouse embryonic stem cells}, volume={42}, number={6}, journal={Journal of Animal Science}, author={Lee, C. K. and Piedrahita, J. A.}, year={2001}, pages={941–956} }
 @article{barthel_feng_piedrahita_mcmurray_templeton_adams_2001, title={Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival}, volume={69}, DOI={10.1128/IAI.69.5.3110-3119.2001}, abstractNote={ABSTRACT}, number={5}, journal={Infection and Immunity}, author={Barthel, R. and Feng, J. and Piedrahita, Jorge and McMurray, D. N. and Templeton, J. W. and Adams, L. G.}, year={2001}, pages={3110–3119} }
 @article{miller_kennedy_thomson_han_smith_ing_piedrahita_busbee_2000, title={A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity}, volume={55}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/55.1.69}, abstractNote={A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.}, number={1}, journal={TOXICOLOGICAL SCIENCES}, author={Miller, S and Kennedy, D and Thomson, J and Han, F and Smith, R and Ing, N and Piedrahita, J and Busbee, D}, year={2000}, month={May}, pages={69–77} }
 @article{lee_piedrahita_2000, title={Effects of growth factors and feeder cells on porcine primordial germ cells in vitro}, volume={2}, DOI={10.1089/152045500454753}, abstractNote={As embryonic stem (ES) cells are not available in swine, embryonic germ (EG) cells derived from primordial germ cells (PGCs) are an alternate source of pluripotent embryonic cells for genetic modification through homologous recombination. Although morphological and biochemical characteristics are similar between ES and EG cells, culture conditions are quite different. To optimize the culture condition for the establishment of porcine EG cells, porcine PGCs were cultured in vitro with various combinations of growth factors (leukemia inhibitory factor [LIF], stem cell factor [SCF], and basic fibroblast growth factor [bFGF]) and on different kinds of feeder cells (STO, TM(4), Sl/Sl(4) m220, porcine embryonic fibroblasts, and COS-7 cells). Optimal results were obtained when all three growth factors (LIF, SCF, and bFGF) were present in the media. Also, feeder cells expressing membrane-bound SCF are required for survival and establishment of porcine EG cells. Therefore, a combination of growth factors and proper feeder cells are critical for the establishment of undifferentiated porcine EG cells.}, journal={Cloning and Stem Cells}, author={Lee, C-K and Piedrahita, Jorge}, year={2000}, pages={197–206} }
 @article{lee_weaks_johnson_bazer_piedrahita_2000, title={Effects of protease inhibitors and antioxidants on in vitro survival of porcine primordial germ cells}, volume={63}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod63.3.887}, abstractNote={Abstract One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors, and antioxidants on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, α2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (P < 0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of α2-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.}, number={3}, journal={BIOLOGY OF REPRODUCTION}, author={Lee, CK and Weaks, RL and Johnson, GA and Bazer, FW and Piedrahita, JA}, year={2000}, month={Sep}, pages={887–897} }
 @article{piedrahita_2000, title={Gene targeting in domestic species: A new beginning}, volume={9}, ISSN={0962-8819}, DOI={10.1023/A:1008984711570}, number={4/5}, journal={Transgenic Res}, author={Piedrahita, J.A.}, year={2000}, pages={261–262} }
 @article{lee_moore_scales_westhusin_newton_im_piedrahita_2000, title={Isolation and genetic transformation of primordial germ cell (PGC)-derived cells from cattle, goats, rabbits and rats}, volume={13}, ISSN={["1976-5517"]}, DOI={10.5713/ajas.2000.587}, number={5}, journal={ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES}, author={Lee, CK and Moore, K and Scales, N and Westhusin, M and Newton, G and Im, KS and Piedrahita, JA}, year={2000}, month={May}, pages={587–594} }
 @article{barthel_piedrahita_mcmurray_payeur_baca_guemes_perumaalla_ficht_templeton_adams_2000, title={Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis}, volume={61}, ISSN={["1943-5681"]}, DOI={10.2460/ajvr.2000.61.1140}, abstractNote={Abstract}, number={9}, journal={AMERICAN JOURNAL OF VETERINARY RESEARCH}, author={Barthel, R and Piedrahita, JA and McMurray, DN and Payeur, J and Baca, D and Guemes, FS and Perumaalla, VS and Ficht, TA and Templeton, JW and Adams, LG}, year={2000}, month={Sep}, pages={1140–1144} }
 @article{westhusin_piedrahita_2000, title={Three little pigs worth the huff and puff?}, volume={18}, ISSN={1087-0156 1546-1696}, url={http://dx.doi.org/10.1038/81119}, DOI={10.1038/81119}, number={11}, journal={Nature Biotechnology}, publisher={Springer Nature}, author={Westhusin, Mark and Piedrahita, Jorge}, year={2000}, month={Nov}, pages={1144–1145} }
 @article{piedrahita_oetama_bennett_waes_lacey_kamen_richardson_lark_finnell_1999, title={Inactivation of the folate binding protein genes disrupts neural tube closure.}, volume={23}, journal={Nature Genetics}, author={Piedrahita, J. A. and Oetama, B. and Bennett, G. and Waes, J. V. and Lacey, S. W. and Kamen, B. and Richardson, J. and Lark, R. and Finnell, R.}, year={1999}, pages={228–232} }
 @article{ramsoondar_christopherson_guilbert_dixon_ghahary_ellis_wegmann_piedrahita_1999, title={Regulated Expression of MHC class I molecules in the porcine embryo}, volume={60}, journal={Biology of Reproduction}, author={Ramsoondar, J. J. and Christopherson, R. J. and Guilbert, L. J. and Dixon, W. T. and Ghahary, A. and Ellis, S. and Wegmann, T. G. and Piedrahita, J. A.}, year={1999}, pages={387–397} }
 @article{atshaves_foxworth_frolov_roths_kier_oetama_piedrahita_schroeder_1998, title={Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion}, volume={274}, ISSN={["0363-6143"]}, DOI={10.1152/ajpcell.1998.274.3.c633}, abstractNote={The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-( N-methyl)- N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant ( Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deffby 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.}, number={3}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY}, author={Atshaves, BP and Foxworth, WB and Frolov, A and Roths, JB and Kier, AB and Oetama, BK and Piedrahita, JA and Schroeder, F}, year={1998}, month={Mar}, pages={C633–C644} }
 @article{vazquez_nogues_rucker_piedrahita_1998, title={Factors affecting the efficiency of introducing precise genetic changes in ES cells by homologous recombination: tag-and-exchange versus the Cre-loxp system}, volume={7}, ISSN={["0962-8819"]}, DOI={10.1023/A:1008888929552}, abstractNote={The introduction of genetic modifications in specific genes by homologous recombination provides a powerful tool for elucidation of structure-function relationships of proteins of biological interest. Presently, there are several alternative methods of homologous recombination that permit the introduction of small genetic modifications in specific loci. Two of the most widely used methods are the tag-and-exchange, based on the use of positive-negative selection markers, and the Cre-loxP system, based on the use of a site-specific recombinase. The efficiency of detection of targeting events at different loci using the two systems was compared. Additionally, we analysed how the distance between two gene markers placed within the region of homology of a targeting vector affects the rate at which both markers are introduced into the locus during the homologous recombination event. Our results indicate that the method based on the use of positive-negative selection markers was less efficient than the Cre-loxP based system, irrespective of locus or type of positive-negative selection. It was also determined that as the distance between the selectable marker and the genetic modification being introduced increases, there is a progressive reduction in the efficiency of detecting events with the desired genetic modification.}, number={3}, journal={TRANSGENIC RESEARCH}, author={Vazquez, JC and Nogues, C and Rucker, EB and Piedrahita, JA}, year={1998}, month={May}, pages={181–193} }
 @article{piedrahita_moore_oetama_lee_scales_ramsoondar_bazer_ott_1998, title={Generation of transgenic porcine chimeras using primordial germ cell-derived colonies}, volume={58}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod58.5.1321}, abstractNote={In mice, two pluripotent cell lines, embryonic stem (ES) cells and embryonic germ (EG) cells, have been identified. We present here results indicating that porcine EG cell lines can be isolated, genetically transformed, and utilized to make transgenic chimeras. Briefly, primordial germ cells (PGCs) were isolated from Day 25-27 fetuses and plated on STO feeder cells in Dulbecco's modified Eagle's medium:Ham's F-10 medium supplemented with 0.01 mM nonessential amino acids, 2 mM glutamine, 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 40 ng/ml human stem cell factor, 20 ng/ml human basic fibroblast growth factor, and 20 ng/ml human leukemia inhibitory factor. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein under control of the cytomegalovirus promoter. After electroporation, cells were plated and later examined under fluorescein isothiocyanate excitation. Fluorescent colonies were selected for chimera generation. Blastocysts collected from gilts on Day 5 were injected with 10-15 transgenic PGC-derived cells and transferred into recipient gilts. Gilts were hysterectomized on Day 25, and fetal tissues were analyzed by Southern blotting. Three chimeras out of 20 fetuses analyzed were transgenic. Additionally, when one recipient gilt was allowed to go to term, one piglet with transgenic contribution was identified.}, number={5}, journal={BIOLOGY OF REPRODUCTION}, author={Piedrahita, JA and Moore, K and Oetama, B and Lee, CK and Scales, N and Ramsoondar, J and Bazer, FW and Ott, T}, year={1998}, month={May}, pages={1321–1329} }
 @article{ramsoondar_vazques_rucker_dickson_lunney_piedrahita_1998, title={Isolation, characterization, and polymorphism detection of the porcine apolipoprotein E gene}, volume={29}, DOI={10.1046/j.1365-2052.1998.00273.x}, abstractNote={The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (ape‐E) gene. A single positive recombinant phage clone containing a 10·7‐kb insert was isolated from a porcine genomic library, and a 4·2‐kb fragment was subcloned and sequenced. The 4·2‐kb fragment contained the entire apo‐E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo‐E gene is made up of four exons and three introns, and encodes a preapo‐E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo‐E gene contains a (CG)13 microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo‐E gene to chromosome 6 band q2·1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.}, journal={Animal Genetics}, author={Ramsoondar, J. and Vazques, J. C. and Rucker, E. and Dickson, R. and Lunney, J. and Piedrahita, Jorge}, year={1998}, pages={43–47} }
 @article{rucker_piedrahita_1997, title={Cre-mediated site-directed insertions at the mouse whey acidic protein (mWAP) locus}, volume={48}, DOI={10.1002/(sici)1098-2795(199711)48:3<324::aid-mrd4>3.3.co;2-q}, abstractNote={The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4. Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4). Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette. Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion. Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus. The frequency of this event was 23% (11 of 48) of that obtained with random integration. This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells. Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells. Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal “bioreactor” models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements. In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner. It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland. Mol. Reprod. Dev. 48:324–331, 1997. © 1997 Wiley-Liss, Inc.}, journal={Molecular Reproduction and Development}, author={Rucker, E. and Piedrahita, Jorge}, year={1997}, pages={324–331} }
 @article{moore_piedrahita_1997, title={Effect of LIF and culture media on the in vitro differentiation of isolated porcine inner cell masses}, volume={33}, journal={In Vitro Cellular & Developmental Biology. Animal}, author={Moore, K. and Piedrahita, J. A.}, year={1997}, pages={61–70} }
 @article{piedrahita_weaks_petrescu_shrode_derr_womack_1997, title={Genetic characterization of the bovine leukaemia inhibitory factor (LIF) gene: Isolation and sequencing, chromosome assignment and microsatellite analysis}, volume={28}, ISSN={["0268-9146"]}, DOI={10.1111/j.1365-2052.1997.00054.x}, abstractNote={The bovine leukaemia inhibitory factor was isolated from a phage library and sequences for the gene, in addition to 1213bp of 5' and 432bp of 3' sequences, were obtained and compared with other mammalian leukaemia inhibitory factor genes. Comparisons indicated amino acid homologies ranging from 89·6% to 77·2% with the human and mouse homologues, respectively. Analysis of 500bp of 5' regulatory regions indicated homologies ranging from 83·6% to 74·4% with the corresponding human and sheep sequences, respectively. Additionally, bovine leukaemia inhibitory factor‐specific primers were prepared, and a panel of bovine × hamster somatic cell lines were analysed by the polymerase chain reaction (PCR). Data indicated 93% concordance of leukaemia inhibitory factor with aldehyde dehydrogenase 2 located on bovine chromosome 17, and concordance of 81% with myelin basic protein situated on bovine chromosome 24. Southern analysis of selected hybrids confirmed the PCR results, thus conclusively assigning the bovine leukaemia inhibitory factor gene to chromosome 17. Sequence analysis also revealed a microsatellite in intron 2 of the bovine leukaemia inhibitory factor. Analysis of this region by PCR in 22 unrelated Bos taurus and 19 unrelated Bos indicus cattle detected nine different alleles. Polymorphic information content values were 0·53 and 0·80 in B. taurus and B. indicus, respectively. Additionally, the same leukaemia inhibitory factor primers successfully detected allelic variants at this locus in Bos javanicus, Bos guarus and Bison bison but not in Odocoileus virginianus.}, number={1}, journal={ANIMAL GENETICS}, author={Piedrahita, JA and Weaks, R and Petrescu, A and Shrode, TW and Derr, JN and Womack, JE}, year={1997}, month={Feb}, pages={14–20} }
 @article{weaks_ramsoondar_gallager_nogues_piedrahita_1997, title={Isolation, characterization, and chromosome assignment of the porcine ciliary neurotrophic factor}, volume={28}, journal={Animal Genetics}, author={Weaks, R. and Ramsoondar, J. and Gallager, D. and Nogues, C. and Piedrahita, J. A.}, year={1997}, pages={354–357} }
 @article{piedrahita_moore_lee_weaks_ramsoondar_oetama_vasquez_1997, title={Progress on the generation of transgenic pigs via cultured cells}, volume={52}, journal={Journal of Reproduction & Fertility}, author={Piedrahita, J. A. and Moore, K. and Lee, C. and Weaks, R. and Ramsoondar, J. and Oetama, B. and Vasquez, J.}, year={1997}, pages={245–254} }
 @article{finnell_wlodarczyk_craig_piedrahita_bennett_1997, title={Strain dependent alterations in the expression of folate pathway genes following teratogenic exposure to valproic acid in a mouse model.}, volume={70}, DOI={10.1002/(sici)1096-8628(19970613)70:3<303::aid-ajmg17>3.3.co;2-a}, abstractNote={The molecular basis for the well-established hierarchy of susceptibility to valproic acid-induced neural tube defects in inbred mouse strains was examined using in situ transcription and anti-sense RNA amplification methodologies with both univariate and multivariate analyses of the resulting gene expression data. The highly sensitive SWV strain demonstrated a significant reduction in the expression of the folate binding protein (FBP-1) following the teratogenic insult at gestational day 8:18, while the more resistant LM/Bc embryos were up-regulating this gene in response to valproic acid treatment. More importantly, at all 3 gestational timepoints spanning the period of murine neural tube closure examined in this study, the LM/Bc embryos had significantly higher MTHFR (5,10-methylenetetra-hydrofolate reductase) gene expression levels compared to the SWV embryos. As this folate pathway enzyme is important in homocysteine and methionine metabolism, it suggests that the SWV embryos may be hypomethylated, and essential gene expression during critical periods of neural tube closure is compromised by the teratogenic exposure to valproic acid. This study represents the first evidence of a strain difference in transcriptional activity in response to a teratogenic exposure that might be causally related to the development of the teratogen-induced congenital malformations. Am. J. Med. Genet. 70:303–311, 1997. © 1997 Wiley-Liss, Inc.}, journal={American Journal of Medical Genetics. Part A}, author={Finnell, R. H. and Wlodarczyk, B. C. and Craig, J. C. and Piedrahita, Jorge and Bennett, G. D.}, year={1997}, pages={303–311} }
 @article{moore_piedrahita_1996, title={Effect of hematopoetic cytokines on in vitro differentiation of isolated porcine inner cell masses}, volume={45}, journal={Molecular Reproduction and Development}, author={Moore, K. and Piedrahita, J. A.}, year={1996}, pages={139–144} }
 @article{sapatino_petrescu_rosenbaum_smith_piedrahita_welsh_1995, title={CHARACTERISTICS OF CLONED CEREBROVASCULAR ENDOTHELIAL-CELLS FOLLOWING INFECTION WITH THEILERS VIRUS .2. PERSISTENT INFECTION}, volume={62}, ISSN={["0165-5728"]}, DOI={10.1016/0165-5728(95)00094-4}, abstractNote={Cloned cerebrovascular endothelial cells (CVE) persistently infected with Theiler's virus (PI-CVE) have been established and characterized. The CVE were derived from strains of mice that are susceptible (SJL/J and CBA) and resistant (BALB/c) to Theiler's virus-induced demyelination (TVID). The cells were persistently infected with either the BeAn or GDVII strains of Theiler's virus in vitro and studied at various passage levels for infectious virus, viral antigen and the expression of major histocompatibility complex (MHC) Class I and II antigens. The virus replicated to lower titers than in acutely infected CVE and appeared to be more cell-associated. Flow cytometric analysis revealed that 18–39% of the PI-CVE contained viral antigen. Persistently infected CVE derived from SJL/J and CBA mice expressed high levels of MHC Class I, whereas BALB/c PI-CVE did not. MHC Class II was upregulated by IFN-γ in SJL/J PI-CVE albeit at a slightly lower level than in uninfected CVE. In addition, the PI-CVE demonstrated increased levels of mRNA for IL-1β when compared to uninfected CVE.}, number={2}, journal={JOURNAL OF NEUROIMMUNOLOGY}, author={SAPATINO, BV and PETRESCU, AD and ROSENBAUM, BA and SMITH, R and PIEDRAHITA, JA and WELSH, CJR}, year={1995}, month={Nov}, pages={127–135} }
 @article{piedrahita_zhang_hagaman_oliver_maeda_1992, title={GENERATION OF MICE CARRYING A MUTANT APOLIPOPROTEIN-E GENE INACTIVATED BY GENE TARGETING IN EMBRYONIC STEM-CELLS}, volume={89}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.89.10.4471}, abstractNote={We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, pJPB63 and pNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.}, number={10}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={PIEDRAHITA, JA and ZHANG, SH and HAGAMAN, JR and OLIVER, PM and MAEDA, N}, year={1992}, month={May}, pages={4471–4475} }
 @article{piedrahita_gillespie_maeda_1992, title={PRODUCTION OF CHIMERIC HAMSTERS BY AGGREGATION OF 8-CELL EMBRYOS}, volume={47}, ISSN={["0006-3363"]}, DOI={10.1095/biolreprod47.3.347}, abstractNote={Chimeric animals were produced by aggregation of 8-cell-stage embryos from two strains of hamsters (LVG and Bio 1.5). Two series of experiments were performed. In the first series, embryo pairs in contact with each other were classified as aggregates even if 2 distinct embryos could still be distinguished. Of 88 aggregates transferred, 2 chimeras were obtained. Pregnancy rate was 25%, and embryo survival was 35%. In the second set of experiments, only embryo pairs that had coalesced to form a single giant blastocyst were classified as aggregates. Of 56 aggregates transferred, 6 chimeras were obtained. Pregnancy rate was 83%, and embryo survival was 30%. Of the 8 chimeras, 6 were phenotypic males, and 2 were phenotypic females. Both females were germ line chimeras. Of the 6 males, 4 reproduced normally, 1 had abnormal external genitalia but normal spermatogenesis, and 1 was sterile and had atrophic testes. Each of the fertile males transmitted only a single component, either the LVG or the Bio 1.5. Examination of the testes from the sterile chimera revealed that in excess of 80% of the seminiferous cords were devoid of germ cells. These results demonstrate that hamster chimeras can be obtained by aggregation of 8-cell-stage embryos.}, number={3}, journal={BIOLOGY OF REPRODUCTION}, author={PIEDRAHITA, JA and GILLESPIE, L and MAEDA, N}, year={1992}, month={Sep}, pages={347–354} }
 @article{zhang_reddick_piedrahita_maeda_1992, title={SPONTANEOUS HYPERCHOLESTEROLEMIA AND ARTERIAL LESIONS IN MICE LACKING APOLIPOPROTEIN-E}, volume={258}, ISSN={["1095-9203"]}, DOI={10.1126/science.1411543}, abstractNote={Apolipoprotein E (apoE) is a ligand for receptors that clear remnants of chylomicrons and very low density lipoproteins. Lack of apoE is, therefore, expected to cause accumulation in plasma of cholesterol-rich remnants whose prolonged circulation should be atherogenic. ApoE-deficient mice generated by gene targeting were used to test this hypothesis and to make a mouse model for spontaneous atherosclerosis. The mutant mice had five times normal plasma cholesterol, and developed foam cell-rich depositions in their proximal aortas by age 3 months. These spontaneous lesions progressed and caused severe occlusion of the coronary artery ostium by 8 months. The severe yet viable phenotype of the mutants should make them valuable for investigating genetic and environmental factors that modify the atherogenic process.}, number={5081}, journal={SCIENCE}, author={ZHANG, SH and REDDICK, RL and PIEDRAHITA, JA and MAEDA, N}, year={1992}, month={Oct}, pages={468–471} }
 @article{piedrahita_anderson_bondurant_1990, title={INFLUENCE OF FEEDER LAYER TYPE ON THE EFFICIENCY OF ISOLATION OF PORCINE EMBRYO-DERIVED CELL-LINES}, volume={34}, ISSN={["1879-3231"]}, DOI={10.1016/0093-691X(90)90558-B}, abstractNote={Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.}, number={5}, journal={THERIOGENOLOGY}, author={PIEDRAHITA, JA and ANDERSON, GB and BONDURANT, RH}, year={1990}, month={Nov}, pages={865–877} }
 @article{piedrahita_anderson_bondurant_1990, title={ON THE ISOLATION OF EMBRYONIC STEM-CELLS - COMPARATIVE BEHAVIOR OF MURINE, PORCINE AND OVINE EMBRYOS}, volume={34}, ISSN={["1879-3231"]}, DOI={10.1016/0093-691X(90)90559-C}, abstractNote={The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells.}, number={5}, journal={THERIOGENOLOGY}, author={PIEDRAHITA, JA and ANDERSON, GB and BONDURANT, RH}, year={1990}, month={Nov}, pages={879–901} }
 @article{booman_kruijt_tieman_piedrahita_veerhuis_deboer_ruch_1989, title={PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES AGAINST THE H-Y-ANTIGEN}, volume={15}, ISSN={["0165-0378"]}, DOI={10.1016/0165-0378(89)90011-9}, abstractNote={Monoclonal antibodies against the H-Y antigen were produced using spleen cells from female C57BL/6 mice hyperimmunized with cells from syngeneic males. Anti-H-Y positive clones were detected by enzyme immunoassays. Supernatant fluids from Daudi cell cultures and testicular cell preparations taken from mice, rabbits or calves served as presumptive sources of H-Y antigen. In addition, testis supernatant from genetically sterile mice was used. Male specificity was ascertained by the fact that the antibodies could be absorbed with spleen cells from male but not from female mice. Binding of the antibodies to H-Y antigen on the surface of male and female cells, obtained from a number of tissues and species, was confirmed by an indirect immunofluorescence assay. Several monoclonal antibodies appeared to be positive in all assays tested, suggesting that the molecule conferring the H-Y antigenicity lacks species-specificity and appears to be identical for soluble and membrane-bound H-Y antigen.}, number={3}, journal={JOURNAL OF REPRODUCTIVE IMMUNOLOGY}, author={BOOMAN, P and KRUIJT, L and TIEMAN, M and PIEDRAHITA, JA and VEERHUIS, R and DEBOER, P and RUCH, FE}, year={1989}, month={Jul}, pages={195–205} }
 @article{piedrahita_anderson_1985, title={Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos}, volume={74}, DOI={10.1530/jrf.0.0740637}, abstractNote={H-Y antisera were produced in C57BL/6 female mice by repeated intraperitoneal injections of syngeneic male spleen cells. Epididymal spermatozoa were incubated in the presence of H-Y antisera and guinea-pig serum as a complement source. Levels of ATP remaining after treatment were used to calculate the amount of specific killing. Sera of different cytotoxic titres were used in an indirect immunofluorescent assay with a fluorescein isothiocyanate-conjugated IgG fraction of goat anti-mouse IgG (Fc fragment specific) as second antibody. Embryos were classified as fluorescent or nonfluorescent, transferred to pseudopregnant recipients, and allowed to develop to term. Of 12 sera tested for sperm cytotoxicity, 5 were different from a nonimmunized control serum (P less than 0.05). Percentage specific killing in each of these sera was 7.8 +/- 4.2, 11.7 +/- 3.0, 26.0 +/- 2.2, 27.7 +/- 3.7 and 39.2 +/- 4.8, respectively (mean +/- s.e.m. with three replicates). The 5 sera and an additional one (4.9 +/- 1.3% specific killing) were used in the embryo sexing experiment. The accuracy with which these sera correctly identified sex of preimplantation embryos was 60, 46, 74, 73, 74 and 48%, respectively. Correlation coefficients were 0.86 (P less than 0.05) for specific sperm cytotoxicity and percentage of nonfluorescent embryos that were female and 0.78 (n.s.) for specific sperm cytotoxicity and percentage of fluorescent embryos that were male. Therefore, although the sperm cytotoxicity test is useful for screening antisera for the study of H-Y antigen expression on preimplantation embryos, nonfluorescent embryos are more accurately classified as females than are fluorescent embryos as male.}, journal={Journal of Reproduction & Fertility}, author={Piedrahita, Jorge and Anderson, G. B.}, year={1985}, pages={637–644} }