中山大学学报自然科学版 ›› 2018, Vol. 57 ›› Issue (5): 115-122.

• 论文 • 上一篇    下一篇

利用CRISPR/Cas9技术敲除斑马鱼核受体PXR基因初探

成凯1,冯永永2,周莉2,李凯彬2,聂湘平1   

  1. 1. 暨南大学生命科学技术学院生态系//水生生物研究所,广东 广州510632;
    2. 中国水产科学研究院珠江水产研究所,广东 广州510380
  • 收稿日期:2018-03-30 出版日期:2018-09-25 发布日期:2018-09-25
  • 通讯作者: 聂湘平(1966年生),男;研究方向:生态毒理学;E-mail:txpnie@jnu.edu.cn 李凯彬(1973年生),男;研究方向:水生生物学;E-mail:likaibins@126.com

Knocking out nuclear transcriptional factor PXR gene in zebrafish via CRISPR/Cas9 technology

CHENG Kai1, FENG Yongyong2, ZHOU Li2, LI Kaibin2, NIE Xiangping1   

  1. 1. Department of Ecology// Hydrobiology Research Institute, Jinan University,Guangzhou 510632,China;
    2. Pearl River Fisheries Research Institute CAFS, Guangzhou 510380, China  
  • Received:2018-03-30 Online:2018-09-25 Published:2018-09-25

摘要:

孕烷X受体(Pregnane X receptor, PXR)是核受体超家族(NRs)中NR1I的重要成员之一,在保护机体免于内源性和外源性物质损伤方面具有重要作用。利用CRISPR/Cas9基因编辑技术建立PXR基因敲除的斑马鱼模型,为研究环境污染物的毒性机理及代谢过程提供基础模型。根据PXR基因序列,设计gRNA靶位点,体外转录合成gRNA。同时,将设计合成的gRNA与Cas9 mRNA通过显微注射转入斑马鱼受精卵,经孵化、筛选出有效的突变体,并逐代培养杂交筛选出PXR基因敲除突变体。结果表明利用CRISPR/Cas9基因编辑技术成功敲除PXR基因,经测序分析获得稳定的PXR(+4/+4)基因纯合体。

关键词: PXR, CRISPR/Cas9, 斑马鱼

Abstract:

Pregnane X receptor (PXR) transcriptional factor is one of the nuclear receptors family members, which plays a crucial role in the process of the transformation and metabolism of exogenous and endogenerous substances. The present study  focus on the establishment of zebrafish model with PXR gene null by use of CRISPR/Cas9 gene editing technology to provide a fundamental model for the studying of environmental toxicoloical analysis. Target sites of gRNA are designed and synthesized based on the sequence of PXR gene. The mixtures of gRNA and Cas9 mRNA were injected into zebrafish fertilized eggs during single cell phase via microinjection technical approach. Mutant with PXR gene null species were selected out through generational hybridization. Result showed that PXR gene in zebrafish was successfully knocked out by CRISPR/Cas9 technological approach, and sequence analysis exhibited 4 bp insert in the encoding PXR gene region.

Key words: pregnane X receptor(PXR), CRISPR/Cas9, zebrafish

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