Generation of Trpv4 conditional knockout mice

Trpv4^−/− mice were generated using AK7 mouse embryonic stem cells. The Trpv4 gene was targeted by homologous recombination with a construct from the International Knockout (KO) Mice Consortium that bears loxP sites flanking the sixth exon (ID: 79331) (49). After selection of several correctly targeted clones, we injected them into C57BL/6J mice host blastocysts. The embryos were transferred into pseudopregnant CD1 mice. Chimeric male offspring were selected on the basis of the coat color. They were mated with C57BL/6J females to assess the transmission of the targeted allele. Mice harboring the targeted allele were mated with ROSA-Flp females to excise the selection cassette. The resulting mice had the sixth exon of the Trpv4 gene flanked with loxP sites (Trpv4lox). Trpv4^lox/lox mice were crossed with a mouse line expressing the Cre recombinase under the Phosphoglycerate kinase 1 (PGK-1) promoter to obtain a constitutive Trpv4 KO mouse line (Trpv4^−/−). Loss of expression of TRPV4 in Trpv4^−/− mice was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), by Western blotting on kidney or PT extracts, and by immunohistochemistry (Fig. 1, A to C).

To obtain the conditional Trpv4 KO mouse model, mice with the Trpv4lox allele were bred with Trpv4^−/− mice heterozygous for the ROSA26-lox-stop-lox-tdTomato reporter gene to obtain the Trpv4^lox/− Tom^lox genotype. To achieve deletion specifically in PTs, Trpv4^lox/− mice were crossed with mice expressing the Cre fusion protein under the control of the regulatory elements of the PEPCK gene (PEPCK-Cre transgene) (43). Heterozygous transgenic mice Trpv4^lox/− Tom^lox/− PEPCK-Cre^+/− and their Trpv4^lox/− Tom^lox/− PEPCK-Cre^−/− littermates were identified by PCR genotyping.