Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.
J Reprod Dev
; 61(5): 449-57, 2015.
Article
em En
| MEDLINE
| ID: mdl-26227017
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Animais Geneticamente Modificados
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Sus scrofa
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Técnicas de Inativação de Genes
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Galactosiltransferases
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Oxigenases de Função Mista
Limite:
Animals
País/Região como assunto:
Asia
Idioma:
En
Revista:
J Reprod Dev
Assunto da revista:
MEDICINA REPRODUTIVA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Japão