基于CRISPR/Cas9技术的Wip1基因敲除ST细胞的建立

doi:10.11843/j.issn.0366-6964.2021.010.012

摘要/Abstract

摘要： 旨在采用CRISPR/Cas9编辑系统获得Wip1基因敲除的猪睾丸（swine testis，ST）细胞，为后续在细胞水平上探究Wip1基因对ST细胞增殖、凋亡的影响奠定基础。本研究将实验室已有的靶向Wip1基因第一外显子的两条sgRNA（sgWip1-1和sgWip1-2）分别连接到pX330-GFP和pX330-RFP载体，然后共转染ST细胞（从80~90日龄的猪胚胎睾丸组织中分离出未成熟的支持细胞）。利用流式细胞仪收集红绿双荧光阳性的ST细胞，以用于单克隆细胞挑选。对得到的30个单克隆细胞进行基因型鉴定，并将PCR产物进行T-A克隆。结果显示，有29个单克隆细胞出现了基因片段敲除，Wip1基因的片段敲除效率为96.7%。其中单等位基因片段敲除的有5个，纯合双等位基因片段敲除的有8个，杂合双等位基因片段敲除的有16个。本研究高效地构建了Wip1基因敲除的ST细胞，不仅为后续研究Wip1基因对ST细胞增殖、凋亡的影响提供了细胞模型，也为研究Wip1基因对公猪繁殖性能的影响奠定了基础。

关键词: CRISPR/Cas9, Wip1基因, ST细胞, 猪

Abstract: The purpose of this study was to develop Wip1-knockout swine testis (ST) cells using CRISPR/Cas9 system, which laid a foundation for furture exploring the roles of Wip1 gene in the proliferation and apoptosis of ST cells. In this study, two sgRNA (sgWip1-1 and sgWip1-2) targeting the first exon of Wip1 gene were respectively cloned into pX330-GFP and pX330-RFP vectors, and then co-transfected into ST cells (immature Sertoli cell isolated from 80-90 day-old pig embryo testes). The positive ST cells with red and green fluorescence were selected by flow cyto-metry to screen monoclonal cells. The 30 monoclonal cells were performed for genotype identification, and then the PCR products were used to implement T-A clone. In total, 29 monoclonal cells were exhibited fragment deletions, including 5 clones with monoallelic fragment deletions, 8 clones with biallelic homozygous fragment deletions, 16 clones with biallelic heterozygous fragment deletions. The fragment knockout efficiency was 96.7%. In conclusion, we efficiently obtained the Wip1-knockout ST cells. This work not only provides a cell model for subsequent researching the effects of Wip1 gene on the proliferation and apoptosis of ST cells, but also lays a good foundation for exploring the roles of Wip1 gene on the reproductive performance of boars.

Key words: CRISPR/Cas9, Wip1 gene, ST cells, pig

中图分类号:

S828.2

车晶晶, 徐奎, 张秀玲, 向光明, 王楠, 王悦, 刘志国, 牟玉莲, 李奎. 基于CRISPR/Cas9技术的Wip1基因敲除ST细胞的建立[J]. 畜牧兽医学报, 2021, 52(10): 2814-2821.

CHE Jingjing, XU Kui, ZHANG Xiuling, XIANG Guangming, WANG Nan, WANG Yue, LIU Zhiguo, MU Yulian, LI Kui. Establishment of Wip1-Knockout ST Cells Mediated by CRISPR/Cas9 System[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2814-2821.

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