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目的利用常间回文重复序列丛集(CRISPR)/CRISPR相关蛋白9(Cas9)系统敲除小鼠肾内髓集合管3(m-IMCD3)上皮细胞的水通道蛋白2(Aqp2)基因。方法在小鼠Aqp2基因的第1、4个外显子上各设计两个小向导RNA(sgRNA),分别为sgRNA1-1、sgRNA1-2、sgRNA4-1、sgRNA4-2,并将其成功克隆进常间回文重复序列丛集慢病毒载体2上。将测序正确的质粒转染到m-IMCD3细胞中,提取各单克隆细胞系的基因组DNA,通过聚合酶链反应扩增出sgRNA作用靶点的DNA片段并测序。利用逆转录聚合酶链反应(RT-PCR)及免疫荧光检测Aqp2的表达。结果成功构建出含有相应sgRNA的载体;4个sgRNA对Aqp2基因的靶点都有切割作用;sgRNA1-1与sgRNA4-2组合能成功把两者间5500bp的DNA片段敲除;应用RT-PCR及免疫荧光证实应用CRISPR/Cas9系统敲除Aqp2后,该m-IMCD3细胞系中的Aqp2mRNA及蛋白几乎未见表达。结论通过CRISPR/Cas9系统可获得Aqp2基因敲除的肾集合管上皮细胞系。
OBJECTIVE: To knock down the aquaporin 2 (Aqp2) gene of mouse renal medulla collecting tube 3 (m-IMCD3) epithelial cells using the CRESPR / CRISPR-associated protein 9 (Cas9) system. Methods Two small directional guide RNAs (sgRNAs) were designed on the first and the fourth exons of mouse Aqp2 gene, which were sgRNA1-1, sgRNA1-2, sgRNA4-1 and sgRNA4-2, respectively, and successfully cloned Into the regular palindrome repeat cluster on lentiviral vector 2. The correct sequencing plasmid was transfected into m-IMCD3 cells. The genomic DNA of each monoclonal cell line was extracted. The DNA fragment of target sgRNA was amplified by polymerase chain reaction and sequenced. Aqp2 expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Results The vector containing the corresponding sgRNAs was successfully constructed. Four sgRNAs cleaved the Aqp2 target. The combination of sgRNA1-1 and sgRNA4-2 successfully knocked out the 5500bp DNA fragments. RT-PCR and RT- Immunofluorescence confirmed that Aqp2 mRNA and protein were almost not expressed in the m-IMCD3 cell line after knockdown of Aqp2 using the CRISPR / Cas9 system. Conclusion Aqp2 knockout renal collecting duct epithelial cell line can be obtained by CRISPR / Cas9 system.