Conditional Knockout of LRRC8A in Myofibroblasts

All the experiments involving the use of animals were approved by the Animal Use and Care Committee of the Fourth Military Medical University, Xi'an, China. The Lrrc8a^flox/flox mice were established by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (Cyagen Biosciences Inc., Guangzhou, China). The exon 3 of the Lrrc8a gene was selected as the conditional KO region and the gRNA target sequences are as follows. gRNA1 (Lrrc8a reverse strand match): TGAGATAGTCCATGTAGCCCTGG. gRNA2 (Lrrc8a forward strand match): CAAAGTCGGGCTTAGATGGGTGG. To establish the conditional myofibroblast-specific LRRC8A knockout (CF-KO) mice, the Lrrc8a^flox/flox mice were crossed with tamoxifen-inducible periostin-Cre (PostnMCM) mice (No. 029645, Jackson Laboratory, Marine, US). At 8-12 wk of age, the Lrrc8a^flox/flox/PostnMCM mice were given a tamoxifen hydrochloride-contained chow diet (400 mg/kg, Harlan Teklad, US) for 28 d, followed by a regular chow diet for another 15 d to ensure the clearance of tamoxifen from the animals 18 (link). Lrrc8a^flox/flox/PostnMCM/tamoxifen mice were the CF-KO, while Lrrc8a^flox/flox/tamoxifen littermates were the wild-type (WT) control.