利用CRISPR/Cas9系统构建水通道蛋白9 基因敲除小鼠

doi:10.16098/j.issn.0529-1356.2022.01.018

解剖学报 ›› 2022, Vol. 53 ›› Issue (1): 126-131.doi: 10.16098/j.issn.0529-1356.2022.01.018

利用CRISPR/Cas9系统构建水通道蛋白9 基因敲除小鼠

程全成樊婧刘怀存丁慧如方璇王建伟陈春花* 张卫光*

北京大学基础医学院人体解剖学教研室，北京 100191

收稿日期:2020-05-26 修回日期:2020-07-03 出版日期:2022-02-06 发布日期:2022-02-06
通讯作者: 陈春花;张卫光 E-mail:zhangwg@bjmu.edu.cn
基金资助:
北京市自然科学基金

Construction of aquaporin 9 gene knockout mice using CRISPR/Cas9 gene editing system

CHENG Quan-cheng FAN Jing LIU Huai-cun DING Hui-ru FANG Xuan WANG Jian-wei CHEN Chun-hua* ZHANG Wei-guang*

Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191

Received:2020-05-26 Revised:2020-07-03 Online:2022-02-06 Published:2022-02-06
Contact: CHEN Chun-hua;ZHANG Wei-guang E-mail:zhangwg@bjmu.edu.cn
Supported by:
Natural Science Foundation of Beijing

摘要/Abstract

摘要：

目的利用CRISPR/Cas9系统敲除小鼠水通道蛋白9(AQP-9)基因，构建稳定敲除AQP-9基因的纯合子AQP-9^-/-小鼠。方法根据CRISPR/Cas9靶点设计原则，在Ensembl数据库上找到AQP-9基因序列的外显子区域，综合分析选定AQP-9-202的公共外显子2，前期在其两边设计7个小向导RNA（sgRNA）靶点，选择合适靶点，将AQP-9敲除。用PCR以及基因测序手段检测基因敲除效果。获得F1代杂合子小鼠后，继而提供10只野生型小鼠（雌雄各5只）进行繁育，以期得到纯合子AQP-9^-/-小鼠。结果前期设计的7个sgRNA靶点基因活性均在95%以上，选择L2和R2作为最终的sgRNA靶点。注射受精卵74枚，移植60枚，得到6只F0代阳性鼠。经过繁育鉴定，最终得到稳定敲除AQP-9基因的纯合子AQP-9^-/-小鼠。结论利用CRISPR/Cas9系统获得全身敲除AQP-9基因且可稳定遗传的纯合子AQP-9^-/-小鼠。

关键词: 水通道蛋白9, 规律间隔成簇短回文重复序列/相关蛋白9, 基因敲除, 基因型鉴定, 小鼠

Abstract:

Objective To construct homozygous aquaporin 9(AQP-9)-/- mice using the CRISPR/Cas9 system. Methods According to the design principle of CRISPR/Cas9 target, the exon region of the AQP-9 gene sequence was found in the Ensembl database. AQP-9-202 exon was selected through comprehensive analysis. In the early stage, 7 small guide RNA(sgRNA) targets were designed on both sides of it, appropriate targets were selected and AQP-9 was knocked out. The knockout result were detected by PCR and gene sequencing. After the F1 heterozygous mice were obtained, 10 wild-type mice (5 males and 5 females) were provided for breeding in order to obtain homozygous AQP-9-/- mice. Results After injection of 74 fertilized eggs and transplantation of 60 pieces, 6 F0 generation positive mice were obtained.After breeding and identification, the homozygous AQP-9-/- mice were finally obtained. Conclusion Homozygous AQP-9-/- mice with stable inheritance could be obtained by using the CRISPR/Cas9 system.

Key words: Aquaporin 9, Clustered regularly interspaced short palindromic repeats/-associated protein-9, Gene knockout, Genotype identification, Mouse

中图分类号:

R319

程全成樊婧刘怀存丁慧如方璇王建伟陈春花张卫光. 利用CRISPR/Cas9系统构建水通道蛋白9 基因敲除小鼠[J]. 解剖学报, 2022, 53(1): 126-131.

CHENG Quan-cheng FAN Jing LIU Huai-cun DING Hui-ru FANG Xuan WANG Jian-wei CHEN Chun-hua ZHANG Wei-guang . Construction of aquaporin 9 gene knockout mice using CRISPR/Cas9 gene editing system[J]. Acta Anatomica Sinica, 2022, 53(1): 126-131.

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利用CRISPR/Cas9系统构建水通道蛋白9 基因敲除小鼠

Construction of aquaporin 9 gene knockout mice using CRISPR/Cas9 gene editing system

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