The Experts below are selected from a list of 2232 Experts worldwide ranked by ideXlab platform
Hiroshi Murakami - One of the best experts on this subject based on the ideXlab platform.
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production of alpha 1 3 galactosyltransferase gene knockout Pigs expressing both human decay accelerating factor and n acetylglucosaminyltransferase iii
Molecular Reproduction and Development, 2005Co-Authors: Yoichi Takahagi, Shuji Miyagawa, Tatsuya Fujimura, Hiroshi Nagashima, Tamotsu Shigehisa, Ryota Shirakura, Hiroshi MurakamiAbstract:Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout Pigs were produced with Transgenic Pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the Transgenic Pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO Pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO Pig fetuses were also used for NT to produce cloned KO Pigs. Two healthy clone Pigs were born. These Pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO Pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original Transgenic Pigs and less than a half levels of the alphaGal epitopes existed in wild-type Pig cells.
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production of α1 3 galactosyltransferase gene knockout Pigs expressing both human decay accelerating factor and n acetylglucosaminyltransferase iii
Molecular Reproduction and Development, 2005Co-Authors: Yoichi Takahagi, Shuji Miyagawa, Tatsuya Fujimura, Hiroshi Nagashima, Tamotsu Shigehisa, Ryota Shirakura, Hiroshi MurakamiAbstract:Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout Pigs were produced with Transgenic Pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the Transgenic Pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO Pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO Pig fetuses were also used for NT to produce cloned KO Pigs. Two healthy clone Pigs were born. These Pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO Pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original Transgenic Pigs and less than a half levels of the αGal epitopes existed in wild-type Pig cells. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.
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remodeling of the major Pig xenoantigen by n acetylglucosaminyltransferase iii in Transgenic Pig
Journal of Biological Chemistry, 2001Co-Authors: Shuji Miyagawa, Yoichi Takahagi, Rie Nakai, Ayako Murase, Souichi Koyota, Masaru Koma, M. Yamada, Katsuyoshi Matsunami, Hiroshi Murakami, Daisuke FukutaAbstract:Abstract We have been successful in generating several lines of Transgenic mice and Pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and Pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III Transgenic Pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated Pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the Transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of Pig heart grafts, using a Pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.
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remodeling of the major Pig xenoantigen by n acetylglucosaminyltransferase iii in Transgenic Pig
Journal of Biological Chemistry, 2001Co-Authors: Shuji Miyagawa, Yoichi Takahagi, Rie Nakai, Ayako Murase, Souichi Koyota, Masaru Koma, M. Yamada, Katsuyoshi Matsunami, Hiroshi Murakami, Daisuke FukutaAbstract:We have been successful in generating several lines of Transgenic mice and Pigs that contain the human beta-d-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and Pigs reduced their antigenicity to human natural antibodies, especially the Galalpha1-3Galbeta1-4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III Transgenic Pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated Pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the Transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of Pig heart grafts, using a Pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.
Hiroshi Nagashima - One of the best experts on this subject based on the ideXlab platform.
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Transgenic Pig expressing the red fluorescent protein kusabira orange as a novel tool for preclinical studies on hepatocyte transplantation
Transplantation proceedings, 2013Co-Authors: Takanobu Shigeta, Hitomi Matsunari, Masahito Watanabe, Kazuhiro Umeyama, Huaiche Hsu, Shin Enosawa, Naoto Matsuno, Mureo Kasahara, Hiroshi NagashimaAbstract:Abstract Introduction Research on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as Pigs. We used Transgenic Pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells. Materials and Methods Donor hepatocytes were isolated from Transgenic Kusabira-Orange (KO)-expressing Pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a Transgenic-cloned KO Pig by Matsunari et al. and ova from a domestic Pig. The hepatocyte transplant recipients were the nonTransgenic, KO-negative littermates. In these recipient Pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the Transgenic male Pig were isolated using collagenase perfusion. Hepatocytes (1 × 10 9 cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient Pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys. Results Strong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the Transgenic male donor Pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient Pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7. Conclusion Transgenic Pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.
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production of Transgenic clone Pigs by the combination of icsi mediated gene transfer with somatic cell nuclear transfer
Transgenic Research, 2006Co-Authors: Mayuko Kurome, Hideto Ueda, Katsutoshi Naruse, Ryo Tomii, Hiroshi NagashimaAbstract:The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce Transgenic Pigs. We also aimed at examining whether Transgenic Pigs can be cloned from somatic cells of a Transgenic Pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into Pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 Transgenic fetuses were obtained. One of the recipients gave birth to three live Piglets including one Transgenic Pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder Transgenic Pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live Transgenic-clones. The transgene copy number and integrity in the founder Pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce Transgenic Pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in Transgenic Pig production.
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production of α1 3 galactosyltransferase gene knockout Pigs expressing both human decay accelerating factor and n acetylglucosaminyltransferase iii
Molecular Reproduction and Development, 2005Co-Authors: Yoichi Takahagi, Shuji Miyagawa, Tatsuya Fujimura, Hiroshi Nagashima, Tamotsu Shigehisa, Ryota Shirakura, Hiroshi MurakamiAbstract:Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout Pigs were produced with Transgenic Pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the Transgenic Pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO Pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO Pig fetuses were also used for NT to produce cloned KO Pigs. Two healthy clone Pigs were born. These Pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO Pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original Transgenic Pigs and less than a half levels of the αGal epitopes existed in wild-type Pig cells. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.
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production of alpha 1 3 galactosyltransferase gene knockout Pigs expressing both human decay accelerating factor and n acetylglucosaminyltransferase iii
Molecular Reproduction and Development, 2005Co-Authors: Yoichi Takahagi, Shuji Miyagawa, Tatsuya Fujimura, Hiroshi Nagashima, Tamotsu Shigehisa, Ryota Shirakura, Hiroshi MurakamiAbstract:Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout Pigs were produced with Transgenic Pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the Transgenic Pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO Pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO Pig fetuses were also used for NT to produce cloned KO Pigs. Two healthy clone Pigs were born. These Pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO Pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original Transgenic Pigs and less than a half levels of the alphaGal epitopes existed in wild-type Pig cells.
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production of a Transgenic Pig expressing human albumin and enhanced green fluorescent protein
Journal of Reproduction and Development, 2005Co-Authors: Katsutoshi Naruse, Hiroshi Nagashima, Hiroshi Ishikawa, Hiroomi Kawano, Hideto Ueda, Mayuko Kurome, Koji Miyazaki, Maiko Endo, Tohru Sawasaki, Masatoshi MakuuchiAbstract:We introduced a fusion gene of human albumin and enhanced green fluorescent protein (EGFP) into porcine oocytes using the sperm vector method, and produced a Piglet that showed clear expression of GFP in the hooves and skin. PCR and Southern blotting analysis of genomic DNA extracted from the Piglet's tissues, including the liver, showed that the tissues carried the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the tissues. The fact that human albumin gene was integrated and expressed in the liver of the Transgenic Pig opened a way for us to achieve our goal, which was the use of Transgenic Pigs for the bioartificial liver support system.
Robert M Petters - One of the best experts on this subject based on the ideXlab platform.
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Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies
PLoS ONE, 2017Co-Authors: Renan B. Sper, Robert M Petters, Sehwon Koh, Xia Zhang, Sean Simpson, Bruce Collins, Jeff Sommer, Ignacio Caballero-posadas, Jeff L. Platt, Jorge A. PiedrahitaAbstract: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-beta 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 beta-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.
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Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies
2017Co-Authors: Renan B. Sper, Robert M Petters, Sehwon Koh, Xia Zhang, Sean Simpson, Bruce Collins, Jeff Sommer, Jeff L. Platt, Ignacio Caballero, Jorge A. PiedrahitaAbstract: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.
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photoreceptor differentiation following transplantation of allogeneic retinal progenitor cells to the dystrophic rhodopsin pro347leu Transgenic Pig
Stem Cells International, 2012Co-Authors: Henry Klassen, Melissa Samuel, Randall S Prather, Fulton Wong, Robert M Petters, Jens Folke Kiilgaard, Karin Warfvinge, M La Cour, M J YoungAbstract:Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the Pig. Methods. Retinal progenitor cells were derived from the neural retina of GFP-Transgenic Pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-Transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous photoreceptor rosettes within GFP-positive grafts, indicative of the development of cellular polarity and self-assembly into rudiments of outer retinal tissue. Conclusion. Together, these data support the tolerance of RPCs as allografts and demonstrate the high level of rod photoreceptor development that can be obtained from cultured RPCs following transplantation. Strategies for further progress in this area, together with possible functional implications, are discussed.
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oxidative damage is a potential cause of cone cell death in retinitis Pigmentosa
Journal of Cellular Physiology, 2005Co-Authors: Jikui Shen, Fulton Wong, Robert M Petters, Xiaoru Yang, Aling Dong, You Wei Peng, Peter A CampochiaroAbstract:Retinitis Pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a Transgenic Pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old Transgenic Pigs in which almost all rods had died, compared to faint staining in two 10-month-old control Pig retinas. In 22- and 24-month-old Transgenic Pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.
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oxidative damage is a potential cause of cone cell death in retinitis Pigmentosa
Journal of Cellular Physiology, 2005Co-Authors: Jikui Shen, Fulton Wong, Robert M Petters, Xiaoru Yang, Aling Dong, You Wei Peng, Peter A CampochiaroAbstract:Retinitis Pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a Transgenic Pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old Transgenic Pigs in which almost all rods had died, compared to faint staining in two 10-month-old control Pig retinas. In 22- and 24-month-old Transgenic Pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation. © 2005 Wiley-Liss, Inc.
D J G White - One of the best experts on this subject based on the ideXlab platform.
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prolonged function of extracorporeal hdaf Transgenic Pig livers perfused with human blood
Transplantation, 2002Co-Authors: Michael Rees, D J G White, Andrew J Butler, Gilda Chavezcartaya, Derek G D Wight, Neil D Casey, Graeme J M Alexander, Sadik A Khuder, P J FriendAbstract:Background. The development of genetically modified Pigs has renewed interest in the use of porcine liver perfusion in the treatment of acute liver failure. Method. A previously developed model of extracorporeal perfusion has been used to test the function of porcine livers Transgenic for human decay accelerating factor when perfused with fresh, whole, human blood. Three experimental groups were studied: alloperfusions (normal Pig livers perfused with Pig blood) and xenoperfusions of both unmodified and Transgenic Pig livers with human blood. All livers were perfused for up to 72 hr. Results. Alloperfusion resulted in the maintenance of good function and histological structure. Stable hemodynamic, synthetic, and metabolic parameters were demonstrated in both unmodified and Transgenic liver xenoperfusions; hyperacute rejection was not seen. In both groups, however, the measured parameters of liver function deteriorated toward the end of the 72 hr perfusion period; deterioration was more marked in the nonTransgenic group. Xenoperfusions were characterized by a progressive and marked decrease in hematocrit of the circulating blood. Histologically, patchy necrosis was noted in both groups and more retained erythrocytes were seen in the sinusoids of nonTransgenic livers, but no other consistent differences were apparent. Conclusions. These studies have demonstrated that porcine liver xenoperfusions can be performed for prolonged periods while maintaining good liver function. The use of organs from animals Transgenic for a human complement regulator protein confers improvement in some measures of liver function. This preclinical model provides evidence that extracorporeal liver xenoperfusion may be effective in temporary liver support for patients in acute liver failure.
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hla specific antibodies in highly sensitized patients can cause a positive crossmatch against Pig lymphocytes
Transplantation, 1998Co-Authors: Craig J Taylor, D J G White, Kerrie G C Tang, Sheila Smith, Hugh F S DaviesAbstract:Background. The presence of IgG HLA-specific antibodies in the serum of patients awaiting transplantation indicates T- and B-cell priming and would result in acute rejection of a poorly matched human allograft. Recent advances in xenotransplantation, with the amelioration of hyperacute rejection using Transgenic Pig kidneys, may benefit such patients. However, accelerated cellular rejection might result from the primed T-cell recognition of antigenic epitopes shared between Pig and human MHC molecules. Methods. We have compared the reactivity of IgG antibodies from 8 nonsensitized (NS) and 13 highly sensitized (HS) patients with human and Pig lymphocytes by flow cytometry. Xenoreactive natural antibodies (XNA) were absorbed with Pig red blood cells, and HLA class I-specific antibodies were further absorbed with pooled human platelets. Results. Before XNA absorption, 20 of the 21 patients had a positive IgG crossmatch with Pig lymphocytes, and there was no difference between NS and HS patients. In contrast, after XNA absorption, none of the 8 NS patients were positive, compared with 9 of the 13 HS patients (mean of the median channel fluorescence values of 7.7 and 86.5, respectively; P=<0.001). For XNA-absorbed HS patient sera, 20 of 30 (67%) Pig lymphocyte crossmatch combinations were positive, with a mean median channel fluorescence value of 125 (range 31 to 294) compared with 9.5 (range 7 to 13) for the 10 crossmatch-negative combinations. Platelet absorption resulted in a concomitant reduction in antibody binding to Pig lymphocytes in three of six HS patient sera, indicating that HLA class I-specific antibodies are responsible, at least in part, for the positive crossmatch. Conclusion. These results suggest that some IgG HLA-specific antibodies can bind to Pig lymphocytes, analogous to a positive crossmatch with allogeneic donors.
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morphology of hdaf cd55 Transgenic Pig kidneys following ex vivo hemoperfusion with human blood
Transplantation, 1997Co-Authors: M Storck, G Pinochavez, D J G White, D Abendroth, Roland Prestel, Jorg Mullerhoker, C HammerAbstract:Discordant xenotransplantation of Pig kidneys into man may be possible in the future using Transgenic organs which regulate complement activity. It was the aim of this experimental study to characterize morphologic alterations of organs Transgenic for human decay accelerating factor (hDAF/CD55) perfused with human blood since no data on function of these organs after exposure to human blood are available. An ex-vivo system was developed that allows computer driven pressure-controlled perfusion of kidneys including a separate cartridge oxygenator circuit. Following cold ischemia time of 1-4 hr, 8 kidneys from heterozygote Transgenic animals (TG) and 9 control kidneys (C) were perfused with 500 ml freshly drawn heparinized human blood at physiological conditions. A histologic grading system from 0 to +4 was used to describe the histologic findings. Using a mouse antihuman DAF moAB, hDAF was stained on all TG kidneys both on glomerular capillary (4+) and vascular endothelium (2+), but there was no detectable hDAF-expression on controls. No difference in xenoantibody deposition on vascular endothelium was seen between both groups. There was comparable staining for complement fraction C4 in both groups, but significant reduction of C3 and C9 staining on glomerular and vascular endothelium in TG. P-selectin was expressed on a higher level in C (+4) compared with TG (+2). Neutrophil extravasation [NP-57 elastase] was higher in C (80.2 vs. 32.2 C vs. TG [values as n/high power fields). Tubular epithelial cell swelling and mild necrosis was paralleled by glomerular hemorrhage and platelet microthrombus formation in both groups as seen in transmission electron microscopy. The observed results allow the conclusion that hDAF expression on Transgenic Pig kidneys was sufficient to inhibit complement activation beyond C3 during xe-noperfusion with human blood despite xenoantibody deposition.
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prevention of hyperacute rejection by human decay accelerating factor in xenogeneic perfused working hearts
Transplantation, 1996Co-Authors: M Schmoeckel, J Mullerhocker, Georg Nollert, Mehran Shahmohammadi, V K Young, Gilda Chavez, W Kasperkonig, D J G White, Rainer M. Arendt, Ute WilbertlampenAbstract:As a potential source of organs for xenotransplantation, Pigs that are Transgenic for human decay accelerating factor (DAF) have been bred in order to overcome hyperacute rejection. We investigated the protective effect of human DAF in a porcine working heart model perfused by human blood. Hearts of normal landrace Pigs served as controls. The following parameters were measured : stroke work index, coronary flow and arteriovenous oxygen consumption, 6-keto prostaglandin F 1α and prostaglandin E 2 as markers of endothelial cell activation ; creatine phosphokinase and lactate dehydrogenase for evaluation of the extent of myocardial damage ; TNFα and IL-6 as markers of mononuclear cell activation. Histological and ultrastructural investigations from myocardial tissue sections were done at the end of perfusion. Human (h) DAF appeared to inhibit complement-mediated endothelial cell activation of Transgenic Pig hearts successfully. This was in contrast to landrace Pig hearts, which had a sixfold increase of prostaglandin levels during perfusion with human blood. The cardiac weight increase during perfusion time due to interstitial edema tended to be less in the hDAF group. Myocardial damage was minimal in Transgenic hearts, whereas normal Pig hearts produced a threefold increase of creatine phosphokinase and lactate dehydrogenase levels. In these hearts, electron microscopy revealed single cell necrosis of myocytes and vacuolization of mitochondria with cristae rupture. According to the results obtained in the working heart model, the breeding of Pigs that are Transgenic for hDAF represents a promising step to making heart xenotransplantation a clinical reality in the future.
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tissue expression of human complement inhibitor decay accelerating factor in Transgenic Pigs a potential approach for preventing xenograft rejection
Transplantation, 1995Co-Authors: Ariella M Rosengard, G Langford, J Wallwork, N Cary, Alexander W Tucker, D J G WhiteAbstract:Since complement-mediated hyperacute rejection of xenografts prevents the use of Pigs as organ donors to man, the development of Transgenic animals expressing species-specific complement inhibitors could provide a strategy for overcoming hyperacute rejection. The complement inhibitor, human decay-accelerating factor (hDAF), prevents the assembly of C3 and C5 convertases. In this article, the first histologic analysis of hDAF expression in Pig tissues, specifically expression in endothelial cells of Pigs Transgenic for hDAF, is described. Twenty-seven Transgenic Pigs were categorized into 4 groups based on the expression patterns in endothelial, vascular smooth muscle, and squamous epithelial cells of skin biopsy specimens. Skin biopsy specimens permitted evaluation of the Pigs without the need to kill them or to perform invasive procedures. Sixteen cases demonstrated endothelial cell staining. Complete necropsy evaluation, available in 14 of the 27 Pigs, correlated with the skin biopsy specimen expression of hDAF. The immunoperoxidase data matched identically with the presence of the mRNA transcript in 25 of the 26 cases where RNA data were available. Also, the staining patterns of 6 Transgenic Pig founders and their 9 offspring (total of 9 founder-offspring pairs) correlated. Since transgenes are variably expressed in different cell types and since tissue lysates represent a melange of cell types, histologic evaluation for protein expression in tissues from Transgenic animals will be critical if they are to be bred to become clinical organ donors. In addition to endothelial expression of hDAF, its expression on vascular smooth muscle cells may be important in preventing tissue damage when breaks in the endothelium occur.
William H Velander - One of the best experts on this subject based on the ideXlab platform.
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analysis of the n glycans of recombinant human factor ix purified from Transgenic Pig milk
Glycobiology, 2008Co-Authors: Geun Cheol Gil, William H Velander, Kevin E Van CottAbstract:Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the Transgenic Pig mammary gland were determined. The majority of the N-glycans of Transgenic Pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other Transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for Transgenic animal bioreactors.
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purification of recombinant dna derived factor ix produced in Transgenic Pig milk and fractionation of active and inactive subpopulations
Journal of Chromatography A, 2004Co-Authors: Myles Lindsay, William H Velander, Geun Cheol Gil, Armando Cadiz, Chenming Zhang, Kevin E Van CottAbstract:Transgenic animal bioreactors can be engineered to make gram per liter quantities of complex recombinant glycoproteins in milk. However, little is known about the limitations in post-translational processing that occurs for very complex proteins and how this impacts the task of purification. We report on the purification of recombinant factor IX (rFIX) from the milk of Transgenic Pigs having an expression level of 2-3 g rFIX/(l(-1) h(-1)), an expression level that is about 20-fold higher than previously reported. This purification process efficiently recovers highly active rFIX and shows that even complex mixtures like Pig milk, which contains 60 g/l total endogenous milk protein and multiple subpopulations of rFIX, can be processed using conventional, non-immunoaffinity chromatographic methods. Without prior removal of caseins, heparin-affinity chromatography was used to first purify the total population of rFIX at greater than 90% yield. After the total population was isolated, the biologically active and inactive subpopulations were fractionated by high-resolution anion exchange chromatography using an ammonium acetate elution. Capillary isoelectric focusing of the active and inactive rFIX fractions demonstrated that the active subpopulations are the most acidic.
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expression of a functional human complement inhibitor in a Transgenic Pig as a model for the prevention of xenogeneic hyperacute organ rejection
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: William L. Fodor, Barry L. Williams, William H Velander, Louis A Matis, Joseph A Madri, Scott A Rollins, J W Knight, Stephen P SquintoAbstract:The serious shortage of human organs available for transplantation has engendered a heightened interest in the use of animal organs (xenografts) for transplantation. However, the major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection. Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Although endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement, they are species restricted and thus confer limited resistance to activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in xenotransplantation, Transgenic mice and a Transgenic Pig were engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was achieved in a variety of murine and porcine cell types, most importantly on both large vessel and capillary endothelium. hCD59-expressing porcine cells were significantly resistant to challenge with high-titer anti-porcine antibody and human complement. These experiments demonstrate a strategy for developing a Pig-to-primate xenogeneic transplantation model to test whether the expression of a human complement inhibitor in Transgenic Pigs could render xenogeneic organs resistant to hyperacute rejection.
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high level expression of a heterologous protein in the milk of Transgenic swine using the cdna encoding human protein c
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: William H Velander, John L Johnson, R L Page, Christopher G Russell, Anuradha Subramanian, Tracy D Wilkins, Francis C Gwazdauskas, Christoph Pittius, William N DrohanAbstract:Abstract Transgenic Pigs were generated that produced human protein C in their milk at up to 1 g/liter. The gene construct was a fusion gene consisting of the cDNA for human protein C inserted into the first exon of the mouse whey acidic protein gene. These results demonstrate that the mouse whey acidic protein gene contains regulatory elements that can direct cDNA expression at high levels in the Pig mammary gland. Recombinant human protein C that was produced at about 380 micrograms/ml per hr in Transgenic Pig milk possessed anticoagulant activity that was equivalent to that of protein C derived from human plasma. These studies provide evidence that gamma-carboxylation can occur at high levels in the mammary gland of a Pig.