Title: 
Generation of disease model pigs by genome editing

Hiroshi Nagashima 
Meiji University International Institute for Bio-Resource Research

Abstract:
Recent advances in genome editing technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas have enabled the generation of genetically modified pigs with increased efficiency. We have been developing various disease model pigs, including severe combined immunodeficiency, Marfan syndrome, dilated cardiomyopathy, ornithine transcarbamylase deficiency, using genome editing. The efficiency of inducing indels by introducing mRNAs of ZFN or TALEN into porcine primary cultured cells ranged from 6 to 98% depending on the target gene and the genome editing technology used. The gene knockout cells obtained could support embryonic development after nuclear transfer regardless of monoallelic or biallelic knockout. The production efficiencies of the gene knockout cloned-pigs were comparable to those of wild-type and transgenic cloned pigs, indicating that the detrimental effect of gene knockout on embryonic/fetal development was limited. While we have been successfully producing gene knockout pigs using a combination of genome editing technologies and somatic cell cloning, the direct injection of mRNAs of ZFN, TALEN, or CRISPR-Cas into zygotes is also a feasible option. Future prospects for application of gene knockout pigs in biomedical researches will be discussed in this paper.

 

References
Matsunari H, Nagashima H, Watanabe M, Umeyama K, Nakano K, Nagaya M, Kobayashi T, Yamaguchi T, Sumazaki R, Herzenberg LA, Nakauchi H. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc Natl Acad Sci U S A 2013; 110:4557-4562.

Watanabe M, Nakano K, Matsunari H, Matsuda T, Maehara M, Kanai T, Kobayashi M, Matsumura Y, Sakai R, Kuramoto M, Hayashida G, Asano Y, et al. Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA. PLoS One 2013; 8:e76478.