Knockout Of Aquaporin 2 In Mouse Inner Medullary Collecting Duct Cell Line By CRISPR/Cas9 System

Objective To knock out aquaporin 2(Aqp2)in mouse renal inner medullary collecting duct 3( m-IMCD3) epithelial cells by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) system.Methods1. Four small guide RNAs(sgRNA) respectively targeted exon 1 and 4(sgRNA1-1,sgRNA1-2,sgRNA4-1,sgRNA4-2) of Aqp2 gene were designed and successfully ligated to lentiCRISPR V2 vector.2. The plasmid containing a corresponding sgRNA was transfected into m-IMCD3 cells by method of lipofection.3. Cells were filtered by using medium containing 5 ?g/ml puromycin. In addition,when all the cells of the control die, medium containing 1 ?g/ml puromycin was used.4. The genomic DNA of new monoclonal cell lines was extracted.5. A DNA fragment flanked the target site was amplified by genotyping polymerase chain reaction(PCR) with using the template of the genomic DNA of new monoclonal cell lines which was extracted. Then the product of PCR was sequenced.6. The expression of Aqp2 in the cell line was determined by reverse transcriptase polymerase chain reaction(RT-PCR).7. The expression of Aqp2 in the cell line was determined by immunofluorescence.Results1. The lentiCRISPRv2-sgRNA expression vector targeted Aqp2 were successfully constructed.2. The screening cells were successfully acquired.3. The four sgRNAs could cut the Aqp2 gene. The sgRNA1-1 and sgRNA4-2 could successfully knock out 5500 bp DNA fragment.4. Aqp2 mRNA and protein expression in Aqp2 knockout monoclonal cell lines were not detected by RT-PCR and immunofluorescence.Conclusion Aqp2 knockout m-IMCD3 epithelial cells line was established via CRISPR/Cas9 system.