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- W1998370556 abstract "Development of specially designed pigs for basic research and preclinical application of xenotransplantation relies on highly sophisticated animal biotechnologies such as somatic cell cloning and genetic engineering. We have been developing two major technologies, i.e. somatic cell cloning and intracytoplasmic sperm injection (ICSI)-mediated gene transfer, aiming at improving production efficiency of genetically modified pigs. We have produced genetically modified pigs by heterozygous knock-out of α(1,3)-galactosyltransferase gene (GalT-KO) and co-expression of two transgenes, human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GntIII), using the technology of somatic cell nuclear transfer (SCNT) [1]. Cells collected from these pigs were used to generate homozygous GalT-KO cells by means of spontaneous mutation phenomenon of heterozygous GalT-KO cells during cultivation, i.e. loss of heterozygosity. Then homozygous GalT-KO pigs have been successfully cloned by SCNT [2]. Natural breeding of the heterozygous GalT-KO pigs also gave rise to three homozygous GalT-KO pigs. Then we demonstrated that homozygous GalT-KO pigs can be efficiently produced by SCNT as an alternative approach for the preservation of those animals. Primary cultures of preadipocytes [3,4] were established from a homozygous Gal T-KO pig (male) at 6 month old and cryopreserved by routine procedures. Using these cells as nuclear donors and in vitro matured oocytes as recipient cytoplasts, cloned embryos were produced as described previously [5]. Transfer of the 547 cloned embryos into six recipients gave rise to six pregnancies (100%). Although two miscarried at 30 and 21 days of gestation, two recipients farrowed 10 piglets, including two stillborn piglets. From two other recipients a total of seven live full-term piglets were obtained by caesarian section. Overall production efficiency of the cloned piglets was 4.5% (17/378). Homozygous knock-out of the GalT gene was confirmed by PCR and PCR-Southern [1]. A line of our homozygous GalT-KO cells transported to Germany could give rise to cloned offspring by SCNT. The utilization of GalT-KO pigs in clinical applications of xenotransplantation would require further complicated genetic modification, including additional introduction of multiple transgenes to overcome delayed and/or cellular rejections as well as the hyperacute rejection. Serial nuclear transfer is a key technology which enables such “multilayered” genetic modification of pigs. We have therefore been testing the feasibility of serial somatic cell nuclear transfer in pigs. We have so far succeeded in production of fifth generation (G5) cloned pigs. Salivary gland progenitor cells and preadipocytes established from G3 cloned pigs [6] were used as nuclear donors to produce G4 and G5 clones. Production efficiency of clones from G1 to G5 did not differ significantly. Our data demonstrate that G5 cloned pigs can be produced, hence multilayered genetic modification of pigs using serial nuclear transfer is suggested to be a feasible option. In addition, G2 clones with a red fluorescent protein (humanized Kusabira Orange: huKO) gene were produced from a transgenic-cloned pig originating from the same donor cells of the G5 clones. Fibroblast cells derived from the kidney of a transgenic-cloned pig carrying the huKO gene [7] were used to produce the G2 clones. These pigs exhibited the systemic fluorescence expression seen in the original transgenic-cloned pigs. Cloned siblings consisted of the huKO transgenic-cloned pigs and non-transgenic clones originating from the same donor cells would provide a useful model for cell/tissue transplantation experiments. We developed genetically modified pigs as the large animal model for diabetes [8]. Transgenic-cloned pigs carrying a mutant human hepatocyte nuclear factor 1 gene, which is known to cause the type-3 form of maturity-onset diabetes of the young, were produced using a combined technology of ICSI-mediated gene transfer and SCNT. Although most of the 22 cloned offspring obtained died before weaning, four pigs that lived for 20–196 days were diagnosed as diabetes mellitus with non-fasting blood glucose levels greater than 200 mg/dL. Oral glucose tolerance test on a cloned pig also revealed a significant increase of blood glucose level after glucose loading. Histochemical analysis of pancreas tissue from the cloned pigs showed small and irregularly formed Langerhans Islets, in which poor insulin secretion was detected. The present study demonstrated that transgenic-cloned pigs with typical symptom of diabetes can be successfully produced by inducing a dominant-negative mutant using a human mutant gene. References 1. Takahagi Y, Fujimura T, Miyagawa S, et al. Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III. Fmol Reprod Dev 2005; 71: 331–338. 2. Fujimura T, Takahagi Y, Shigehisa T, et al. Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells. Mol Reprod Dev 2008; 75: 1372–1378. 3. Tomii R, Kurome M, Ochiai T, et al. Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes. Cloning Stem Cells 2005; 7: 279–288. 4. Tomii R, Kurome M, Wako N, et al. Production of cloned pigs by nuclear transfer of preadipocytes following cell cycle synchronization by differentiation induction. J Reprod Dev 2009; 55: 121–127. 5. Kurome M, Ueda H, Tomii R, et al. Production of transgenic-clone pigs by the combination of ICSI-mediated gene transfer with somatic cell nuclear transfer. Transgenic Res 2006; 15: 229–240. 6. Kurome M, Hisatomi H, Matsumoto S, et al. Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer. J Reprod Dev 2008; 54: 254–258. 7. Matsunari H, Onodera M, Tada N, et al. Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange. Cloning Stem Cells 2008; 10: 313–324. 8. Umeyama K, Watanabe M, Saito H, et al. Dominant-negative mutant hepatocyte nuclear factor 1alpha induces diabetes in transgenic-cloned pigs. Transgenic Res 2009; 18: 697–706." @default.
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- W1998370556 date "2010-03-01" @default.
- W1998370556 modified "2023-09-27" @default.
- W1998370556 title "Recent advances in production of genetically modified pigs for xenotransplantation" @default.
- W1998370556 doi "https://doi.org/10.1111/j.1399-3089.2010.00573_8.x" @default.
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