Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells

Specific and efficient smooth muscle cell–targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreER(T2)-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreER(T2), is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreER(T2) exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreER(T2)-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLS(P2A) or CreER(T2–P2A) sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLS(P2A)) and inducible (Myh11-CreER(T2–P2A)) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreER(T2)-RAD mice and the Itga8-CreER(T2) mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.