title: Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.
abstract: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.
authors.affiliation: Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsunari
authors.forename: Masahito
authors.initials: M
authors.lastname: Watanabe
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nakano
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Umeyama
authors.forename: Rieko
authors.initials: R
authors.lastname: Sakai
authors.forename: Shuko
authors.initials: S
authors.lastname: Takayanagi
authors.forename: Toki
authors.initials: T
authors.lastname: Takeishi
authors.forename: Tooru
authors.initials: T
authors.lastname: Fukuda
authors.forename: Sayaka
authors.initials: S
authors.lastname: Yashima
authors.forename: Akira
authors.initials: A
authors.lastname: Maeda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Eguchi
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Okuyama
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagaya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D000831:Animals, Newborn; D002460:Cell Line; D019976:Cloning, Organism / Q000662:veterinary; D004624:Embryo Transfer / Q000662:veterinary; D005091:Exons; D005260:Female; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000662:veterinary*; D006720:Homozygote; D007564:Japan; D008297:Male; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; C039919:RNA, recombinant; D012313:RNA; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine