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

doi:10.16098/j.issn.0529-1356.2022.01.018

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Acta Anatomica Sinica ›› 2022, Vol. 53 ›› Issue (1) : 126-131. DOI: 10.16098/j.issn.0529-1356.2022.01.018

Technology and Methodology

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*

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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

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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

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