利用CRISPR/Cas9技术制备成对框基因2敲除小鼠

doi:10.16098/j.issn.0529-1356.2020.04.023

解剖学报 ›› 2020, Vol. 51 ›› Issue (4): 613-617.doi: 10.16098/j.issn.0529-1356.2020.04.023

利用CRISPR/Cas9技术制备成对框基因2敲除小鼠

蔚洪恩¹王敏¹ 赵敏¹石祥呈²李荣山^3*

山西医科大学附属人民医院/山西省人民医院1.神经内科; 2.病理科; 3.肾内科，太原 030012

收稿日期:2019-05-09 修回日期:2019-09-24 出版日期:2020-08-06 发布日期:2020-08-06
通讯作者: 李荣山 E-mail:rongshanli13@163.com
基金资助:
国家自然科学基金面上项目;中国博士后科学基金面上资助项目;山西省青年拔尖人才支持计划;山西省应用基础研究项目

Establishment of paired box gene 2 knockout mice with CRISPR/Cas9 gene targeting technology

WEI Hong-en¹ WANG Min¹ ZHAO Min¹SHI Xiang-cheng² LI Rong-shan^3*

1.Department of Neurology; 2.Department of Pathology; 3.Department of Nephrology, Shanxi Provincial People’s Hospital, Affiliate of Shanxi Medical University, Taiyuan 030012, China

Received:2019-05-09 Revised:2019-09-24 Online:2020-08-06 Published:2020-08-06
Contact: LI Rong-shan E-mail:rongshanli13@163.com

摘要/Abstract

摘要：

目的利用成簇规律间隔短回文重复序列（CRISPR）/重组CRISPR相关核酸酶9（Cas9）技术双切口法制备成对框基因2（Pax2）敲除小鼠，为探讨Pax2基因在多个系统发育的作用提供动物模型。方法根据Pax2基因序列设计sgRNA，设计出的sgRNA和Cas9体外转录后显微注射到C57BL/6J 小鼠的受精卵中，F0代小鼠出生后取其基因DNA测序鉴定基因型。共获得8只F0 代小鼠，使敲除成功的F0代小鼠与野生C57BL/6J 小鼠交配，获得F1代小鼠，后均采用基因成功敲除的小鼠与C57BL/6J 小鼠进行交配，可获得稳定的Pax2基因敲除小鼠。结果成功获得可稳定繁殖的Pax2杂合子基因敲除小鼠，其Pax2基因缺失1628 bp；组织HE染色显示，敲除小鼠的肾小球数量明显减少；Western blotting结果显示，敲除小鼠的肾皮质Pax2蛋白表达较野生型小鼠减少。结论利用CRISPR/Cas9技术可成功构建Pax2杂合子基因敲除小鼠，为进一步研究Pax2基因的作用奠定基础。

关键词: 成对框基因2, CRISPR/Cas9技术, 基因敲除, 肾脏发育障碍, 免疫印迹法, 小鼠

Abstract:

Objective To establish paired box gene 2(Pax2) gene knockout mice by clusterd regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 (Cas9) technology with double nicking method so as to provide an animal model for studying the role of Pax2 gene in multiple development. Methods The sgRNA was designed according to the Pax2 gene sequence, and the designed sgRNA and Cas9 were microinjected into the fertilized eggs of C57BL/6J mice in vitro. F0 generation mice were genetically sequenced to identify genotypes after birth, and a total of 8 F0 generation mice were obtained. The F0 mice were mated with wild C57BL/6J mice to obtain F1 mice, and the mice successfully knocked out were successfully mated with C57BL/6J mice to obtain stable Pax2 knockout mice. Results The stably propagated Pax2 heterozygous knockout mice were successfully obtained, and the Pax2 gene was deleted by 1628 bp. HE staining showed that the number of glomeruli in knockout mice was significantly reduced. Western blotting result showed that the expression of Pax2 protein in renal cortex of knockout mice was reduced compared with that in wild type mice. Conclusion The Pax2 heterozygous knockout mice can be successfully constructed by using CRISPR/Cas9 technology, which lays a foundation for further study of the role of Pax2 gene.

Key words: Paired box gen 2, CRISPR/Cas9 techonology, Gene knockout, Renal developmental disorder, Western blotting, Mouse

中图分类号:

R361⁺1

蔚洪恩王敏赵敏石祥呈李荣山. 利用CRISPR/Cas9技术制备成对框基因2敲除小鼠[J]. 解剖学报, 2020, 51(4): 613-617.

WEI Hong-en WANG Min ZHAO Min SHI Xiang-cheng LI Rong-shan. Establishment of paired box gene 2 knockout mice with CRISPR/Cas9 gene targeting technology[J]. Acta Anatomica Sinica, 2020, 51(4): 613-617.

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利用CRISPR/Cas9技术制备成对框基因2敲除小鼠

Establishment of paired box gene 2 knockout mice with CRISPR/Cas9 gene targeting technology

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