Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

Masahito Watanabe,Yoshikazu Arai,Gota Hayashida,Kazuaki Nakano,Takahiro Kanai,Taisuke Matsuda,Rieko Sakai,Kazuhiro Umeyama,Yoshinori Asano,Yutaka Hanazono,Hitomi Matsunari,Mirina Kobayashi,Momoko Kuramoto,Miki Maehara,Hiroshi Nagashima,Shuko Takayanagi,Masaki Nagaya,Yukina Matsumura,Andrew C Wilber

doi:10.1371/journal.pone.0076478

Masahito Watanabe, Yoshikazu Arai + Show 17 more

Open Access

https://doi.org/10.1371/journal.pone.0076478

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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.

Highlights

Pigs have attracted attention as large experimental animals capable of providing valuable information that is highly extrapolatable to humans due to their anatomical, physiological, and hematological features [1,2,3,4,5]
Design of Zinc finger nuclease (ZFN) and isolation of interleukin-2 receptor gamma (IL2RG) KO cells Similar to IL2RG in humans, mice, and rats, porcine IL2RG is found on the X chromosome and consists of 8 exons [30]
IL2RG KO cells were generated via the electroporation of ZFN-encoding mRNAs into porcine male fetal fibroblasts with transient cold shock treatment at 32uC for 3 d [31]

Summary

Introduction

Pigs have attracted attention as large experimental animals capable of providing valuable information that is highly extrapolatable to humans due to their anatomical, physiological, and hematological features [1,2,3,4,5]. We previously were the first to demonstrate that gene KO in primary porcine fetal fibroblasts in vitro was possible using ZFNs [19], and somatic cells that were genetically modified by ZFNs were shown to be capable of producing gene KO pigs after SCNT [20,21,22,23]. In these studies, the ZFN-encoding plasmid DNA was introduced into somatic cells or the nuclear donor cells for SCNT. We show that an endogenous gene in porcine primary cultured cells could be knocked out using ZFN-encoding mRNAs, thereby allowing the efficient production of a gene KO pig by means of somatic cell cloning

Results

Discussion

Materials and Methods