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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Base Sequence
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Cell Separation
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Cells, Cultured
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Clone Cells
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Deoxyribonucleases / chemistry
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Deoxyribonucleases / metabolism*
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Fibroblasts / cytology
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Fibroblasts / metabolism*
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Gene Knockout Techniques / methods*
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Humans
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Interleukin Receptor Common gamma Subunit / deficiency*
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Interleukin Receptor Common gamma Subunit / genetics*
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Male
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Mice
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Molecular Sequence Data
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Phenotype
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RNA, Messenger / genetics
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Rats
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Swine
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Zinc Fingers*
Substances
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Interleukin Receptor Common gamma Subunit
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RNA, Messenger
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Deoxyribonucleases
Associated data
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GENBANK/AB846644
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GENBANK/AB846645
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GENBANK/AB846646
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GENBANK/AB846647
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GENBANK/AB846648
Grants and funding
This work was supported by the Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency and by the Meiji University International Institute for Bio-Resource Research (MUIIBR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.