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PLoS One. 2013 Oct 9;8(10):e76478. doi: 10.1371/journal.pone.0076478. eCollection 2013.

Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA.

Author information

1
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan ; Meiji University International Institute for Bio-Resource Research (MUIIBR), Kawasaki, Japan.

Erratum in

  • PLoS One. 2014;9(1). doi:10.1371/annotation/99b0e7bf-5878-42de-bcce-719ceea15466.

Abstract

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.

PMID:
24130776
PMCID:
PMC3793986
DOI:
10.1371/journal.pone.0076478
[Indexed for MEDLINE]
Free PMC Article

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