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Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer.
Cheng, Wenmin; Zhao, Heng; Yu, Honghao; Xin, Jige; Wang, Jia; Zeng, Luyao; Yuan, Zaimei; Qing, Yubo; Li, Honghui; Jia, Baoyu; Yang, Cejun; Shen, Youfeng; Zhao, Lu; Pan, Weirong; Zhao, Hong-Ye; Wang, Wei; Wei, Hong-Jiang.
Afiliação
  • Cheng W; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Zhao H; State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
  • Yu H; Research Center of Life Science, Yulin University, Yulin, 719000, China.
  • Xin J; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Wang J; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Zeng L; Hunan Xeno Life Science Co., Ltd, Changsha, 410600, China.
  • Yuan Z; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Qing Y; State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
  • Li H; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Jia B; State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
  • Yang C; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Shen Y; State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
  • Zhao L; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Pan W; State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
  • Zhao HY; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
  • Wang W; State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
  • Wei HJ; Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital Central-South University, Changsha, 410013, China.
Reprod Biol Endocrinol ; 14(1): 77, 2016 Nov 08.
Article em En | MEDLINE | ID: mdl-27821126
ABSTRACT

BACKGROUND:

α1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation.

METHODS:

Transcription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting.

RESULTS:

The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet.

CONCLUSIONS:

TALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Porco Miniatura / Técnicas de Transferência Nuclear / Técnicas de Inativação de Genes / Nucleases dos Efetores Semelhantes a Ativadores de Transcrição / Galactosiltransferases Limite: Animals / Pregnancy Idioma: En Revista: Reprod Biol Endocrinol Ano de publicação: 2016 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Porco Miniatura / Técnicas de Transferência Nuclear / Técnicas de Inativação de Genes / Nucleases dos Efetores Semelhantes a Ativadores de Transcrição / Galactosiltransferases Limite: Animals / Pregnancy Idioma: En Revista: Reprod Biol Endocrinol Ano de publicação: 2016 Tipo de documento: Article