Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts

Ahn, Kwang Sung; Kim, Yong Baek; Kim, Minjeong; Lee, Bo Hyung; Heo, Soon Young; Kang, Man‐Jong; Kang, Yong‐Kook; Lee, Jeong-Woong; Lee, Kyung-Kwang; Kim, Jin‐Hoi; Nho, Whan-Gook; Hwang, Sung Ho; Woo, Jonghye; Park, Jin-Ki; Park, Soo-Bong; Shim, Hosup

doi:10.1016/j.theriogenology.2010.11.001

Cited by 38 publications

(38 citation statements)

References 21 publications

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“…In addition, the methylation and expression patterns did not change in housekeeping genes such as β-actin and GAPDH following serial SCNT. Recently, we and several other research groups successfully generated αGT knock-out pigs by SCNT (Dai et al 2002;Lai et al 2002;Ramsoondar et al 2003) We also recloned piglets by SCNT using somatic cells harvested from postmortem biopsies of the first generation of αGT gene knock-out piglets and successfully resurrected animals with genetic modifications (Ahn et al 2011). However, cloning efficiency by SCNT was exceedingly low.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, we conducted a third SCNT using F2 fibroblast as donors and obtained one piglet (F3). SCNT was carried out as previously described (Ahn et al 2011). Briefly, oocytes were enucleated, and one ear fibroblast cell was transferred into the perivitelline space of an enucleated oocyte in HEPES-buffered TCM-199 supplemented with 0.4% bovine serum albumin (BSA) and 7.5 g/mL cytochalasin B. Fusion of cell-oocyte couplets was accomplished by two direct current pulses (1.5 kV/cm for 40 sec each) in an electrical fusion solution that contained 0.3 M mannitol, 0.5 mM HEPES, 0.05 mM CaCl 2 , and 0.1 mM MgCl 2 .…”

Section: Somatic Cell Nuclear Transfermentioning

confidence: 99%

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Methylation and expression changes in imprinted genesH19andIgf2during serial somatic cell nuclear transfer using piglet fibroblasts

Jang

Kim

et al. 2015

Animal Cells and Systems

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Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the methylation and gene expression patterns. Our study conclusively demonstrates that the methylation patterns of DMRs in H19 and Igf2 were altered, compare to the F0 donor, during serial SCNT, but housekeeping genes were unaffected.

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Section: Discussionmentioning

confidence: 99%

Section: Somatic Cell Nuclear Transfermentioning

confidence: 99%

Methylation and expression changes in imprinted genesH19andIgf2during serial somatic cell nuclear transfer using piglet fibroblasts

Jang

Kim

et al. 2015

Animal Cells and Systems

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“…Porcine ear fibroblasts were prepared from ear skin biopsies from specific pathogen-free (SPF) Minnesota miniature pigs maintained at Seoul National University (Ahn et al, 2011). The cells were cultured with culture medium (DMEM, 15% FBS, 1×non-essential amino acids, 1×sodium pyruvate, 10 −4 M β-mercaptoethanol, 100 unit/ml penicillin, 100 μg/ml streptomycin) for electroporation.…”

Section: Methodsmentioning

confidence: 99%

“…GGTA1 knock-out heterozygous cells (GT-KO) were previously isolated by transfection of GGTA1 knock-out vector into ear fibroblast from miniature pigs (Ahn et al, 2011). The cells were washed with PBS and cultured with 100% human complement serum (Innovative, USA) for 2 h. The serum was replaced with fresh culture medium and cultured for 2 h. Cytotoxicity was detected using CCK-8 kits according to manufacturer’s instruction (Dojindo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus

Kim¹,

Kim²,

Lee³

et al. 2012

Asian Australas. J. Anim. Sci

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The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

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“…The in vitro maturation (IVM) of the oocytes and the SCNT were performed as previously described [20]. Briefly, 42 h after the onset of IVM, the oocytes were enucleated using a glass pipette with a 20-lm internal diameter by aspiration of the first polar body and the second metaphase plate with a small volume of surrounding cytoplasm in HEPES-buffered TCM-199 supplemented with 0.3% bovine serum albumin (BSA) and 5 lg/mL cytochalasin B.…”

Section: Somatic Cell Nuclear Transfermentioning

confidence: 99%

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

Kim

Ahn

Kim

et al. 2014

Biochemical and Biophysical Research Communications

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Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts

Cited by 38 publications

References 21 publications

Methylation and expression changes in imprinted genesH19andIgf2during serial somatic cell nuclear transfer using piglet fibroblasts

Methylation and expression changes in imprinted genesH19andIgf2during serial somatic cell nuclear transfer using piglet fibroblasts

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

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