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

doi:10.16098/j.issn.0529-1356.2022.01.018

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

技术方法

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*

作者信息 +

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*

Author information +

文章历史 +

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。

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.

导出引用

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 https://doi.org/10.16098/j.issn.0529-1356.2022.01.018

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 https://doi.org/10.16098/j.issn.0529-1356.2022.01.018

中图分类号： R319

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