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

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....

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Bibliographic Details
Published in:PloS one 2013-10, Vol.8 (10), p.e76478-e76478
Main Authors: Watanabe, Masahito, Nakano, Kazuaki, Matsunari, Hitomi, Matsuda, Taisuke, Maehara, Miki, Kanai, Takahiro, Kobayashi, Mirina, Matsumura, Yukina, Sakai, Rieko, Kuramoto, Momoko, Hayashida, Gota, Asano, Yoshinori, Takayanagi, Shuko, Arai, Yoshikazu, Umeyama, Kazuhiro, Nagaya, Masaki, Hanazono, Yutaka, Nagashima, Hiroshi
Format: Article
Language:English
Subjects:
Alzheimer's disease
Animal genetic engineering
Animals
Base Sequence
Bovidae
Cell Separation
Cells, Cultured
Clone Cells
Cloning
Cytokines
Deoxyribonucleases - chemistry
Deoxyribonucleases - metabolism
Deoxyribonucleic acid
Diabetes
DNA
DNA binding proteins
Engineering
Fetuses
Fibroblasts
Fibroblasts - cytology
Fibroblasts - metabolism
Gene expression
Gene Knockout Techniques - methods
Genes
Genetic modification
Genetically modified animals
Genome editing
Genomes
Genomics
Hogs
Humans
Interleukin 2
Interleukin Receptor Common gamma Subunit - deficiency
Interleukin Receptor Common gamma Subunit - genetics
Interleukins
Killer cells
Laboratories
Life sciences
Male
Mammals
Messenger RNA
Mice
Molecular Sequence Data
mRNA
Nuclear transfer
Nuclease
Phenotype
Plasmids
Rats
RNA, Messenger - genetics
Rodents
Somatic cell nuclear transfer
Somatic cells
Suidae
Swine
Thymus
Zinc
Zinc finger proteins
Zinc Fingers
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0076478