Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

Shuji MIYAGAWA; Hitomi MATSUNARI; Masahito WATANABE; Kazuaki NAKANO; Kazuhiro UMEYAMA; Rieko SAKAI; Shuko TAKAYANAGI; Toki TAKEISHI; Tooru FUKUDA; Sayaka YASHIMA; Akira MAEDA; Hiroshi EGUCHI; Hiroomi OKUYAMA; Masaki NAGAYA; Hiroshi NAGASHIMA

doi:10.1262/jrd.2015-058

Original Article

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

Shuji MIYAGAWA, Hitomi MATSUNARI, Masahito WATANABE, Kazuaki NAKANO, Kazuhiro UMEYAMA, Rieko SAKAI, Shuko TAKAYANAGI, Toki TAKEISHI, Tooru FUKUDA, Sayaka YASHIMA, Akira MAEDA, Hiroshi EGUCHI, Hiroomi OKUYAMA, Masaki NAGAYA, Hiroshi NAGASHIMA

Author information

Shuji MIYAGAWA
Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
Meiji University International Institute for Bio-Resource Research, Kawasaki 214-8571, Japan
Hitomi MATSUNARI
Meiji University International Institute for Bio-Resource Research, Kawasaki 214-8571, Japan
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Masahito WATANABE
Meiji University International Institute for Bio-Resource Research, Kawasaki 214-8571, Japan
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Kazuaki NAKANO
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Kazuhiro UMEYAMA
Meiji University International Institute for Bio-Resource Research, Kawasaki 214-8571, Japan
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Rieko SAKAI
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Present address: Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
Shuko TAKAYANAGI
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Toki TAKEISHI
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Tooru FUKUDA
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Sayaka YASHIMA
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan
Akira MAEDA
Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
Hiroshi EGUCHI
Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
Hiroomi OKUYAMA
Division of Organ Transplantation, Department of Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
Masaki NAGAYA
Meiji University International Institute for Bio-Resource Research, Kawasaki 214-8571, Japan
Hiroshi NAGASHIMA
Meiji University International Institute for Bio-Resource Research, Kawasaki 214-8571, Japan
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan

Keywords: Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), Double knockout pig, H-D antigen, Transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs)

JOURNAL FREE ACCESS

2015 Volume 61 Issue 5 Pages 449-457

DOI https://doi.org/10.1262/jrd.2015-058

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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.

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