利用CRISPR/Cas9技术构建环指蛋白126基因敲除小鼠

doi:10.3969/j.issn.1674-5817.2019.01.005

实验动物与比较医学 ›› 2019, Vol. 39 ›› Issue (1): 21-25.DOI: 10.3969/j.issn.1674-5817.2019.01.005

利用CRISPR/Cas9技术构建环指蛋白126基因敲除小鼠

高梦樵¹, 艾东旭², 李钰¹, 孙菲¹, 王进¹, 范君文¹, 袁征¹, 刘源¹, 孙兆增¹

1.军事医学研究院实验动物中心, 北京 100071;
2.塔里木大学生命科学学院, 阿拉尔市 843300

收稿日期:2018-10-15 出版日期:2019-02-25 发布日期:2021-01-29
作者简介:高梦樵(1992-),男,硕士研究生,研究方向为实验动物疾病模型。E-mail:1934161039@qq.com
基金资助:
军队实验动物专项[SYDW(2014)006]; 国家传染病重大专项基金(2017ZX10304402-003)

The Construction of Ring Finger Protein 126 Gene Knockout Mouse by Using CRISPR/Cas9 Technique

GAO Meng-qiao¹, AI Dong-xu², LI Yu¹, SUN Fei¹, WANG Jin¹, FAN Jun-wen¹, YUAN Zheng¹, LIU Yuan¹, SUN Zhao-zeng¹

1. Laboratory Animal Center, Academy of Military Medical Sciences, Beijing 100071, China;
2. College of Life Science, Tarim University, Alar 843300, China

Received:2018-10-15 Online:2019-02-25 Published:2021-01-29

摘要/Abstract

摘要： 目的利用CRISPR/Cas9技术,获得环指蛋白126(RNF126)基因敲除小鼠模型。方法针对C57BL/6小鼠Rnf126基因的第四外显子设计敲除引物,构建sgRNA重组表达载体。通过体外转录获得sgRNA及Cas9 mRNA,并以显微注射的方式将RNA导入到C57BL/6小鼠的受精卵中,通过胚胎移植至代孕母鼠。获得子代工程鼠后,用PCR对其进行鉴定。结果成功获得针对鼠Rnf126基因的sgRNA以及Cas9 mRNA; 通过显微注射获得了82枚状态良好的受精卵并成功移植至3只代孕母鼠体内; 获得了11只F0代仔鼠,鉴定后选择一只单链缺失62个碱基的阳性鼠进行扩繁并在F1、F2代检测到该突变。结论成功构建Rnf126基因敲除C57BL/6小鼠,该模型可用于RNF126基因及其表达产物的生物学功能研究。

关键词: CRISPR/Cas9, 环指蛋白126(RNF126), 基因敲除, 动物模型

Abstract: Objective To establish ring finger protein 126 (RNF126) gene knockout mouse model by CRISPR/Cas9 technique and predict the biological function of RNF126 gene. Methods The knockout primers were designed for exon 4 of Rnf126 gene of C57BL/6 mice to construct sgRNA recombinant expression vector. The mixture of sgRNA and Cas9 mRNA , obtained through transcription in vitro, was injected into the fertilized eggs of C57BL/6 mice by microinjection and the fertilized eggs were transferred into surrogate mice. The DNA of offsprings were screened by PCR identification. Results SgRNA for Rnf126 gene and Cas9 mRNA were successfully obtained. A total of 82 fertilized and microinjected zygotes were successfully transferred into 3 surrogate mice. 11 offsprings were obtained in F₀ generation. One of the positive mouse with 62 bases missing as the founder was selected for propagation and the mutation was also detected in F₁ and F₂ generations. Conclusion Rnf126 gene knockout C57BL/6 mouse model was successfully derived, it may be used as a tool for studying the biological function of RNF126 gene.

Key words: CRISPR/Cas9, Ring finger protein 126 (RNF126), Gene knockout, Animal model

中图分类号:

Q95-33

高梦樵, 艾东旭, 李钰, 孙菲, 王进, 范君文, 袁征, 刘源, 孙兆增. 利用CRISPR/Cas9技术构建环指蛋白126基因敲除小鼠[J]. 实验动物与比较医学, 2019, 39(1): 21-25.

GAO Meng-qiao, AI Dong-xu, LI Yu, SUN Fei, WANG Jin, FAN Jun-wen, YUAN Zheng, LIU Yuan, SUN Zhao-zeng. The Construction of Ring Finger Protein 126 Gene Knockout Mouse by Using CRISPR/Cas9 Technique[J]. Laboratory Animal and Comparative Medicine, 2019, 39(1): 21-25.

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利用CRISPR/Cas9技术构建环指蛋白126基因敲除小鼠

The Construction of Ring Finger Protein 126 Gene Knockout Mouse by Using CRISPR/Cas9 Technique

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