Abstract 18828: Generation and Characterization of a Novel Myh11 Knock-In Smooth Muscle Cell-Specific CreEr T2 Mouse Model

Abstract

Background: MYH11 is the most specific and robust marker for differentiated smooth muscle cells (SMCs) and its proximal promoter has been used as a SMC Cre-driver to investigate SMC-specific roles of floxed target genes. Two distinct Myh11-CreER T2 ( Myh11-CreER T2 -Off and -RAD ) mouse lines have been used widely for inducible SMC-specific Cre/loxP-mediated recombination in conditional mutagenesis or lineage tracing. However, both mouse lines were generated using bacterial artificial chromosome (BAC), which were randomly integrated into the genome, potentially resulting in multiple copies of the transgene and the unpredictable disruption of endogenous genes. An additional limitation of the Myh11-CreER T2 -Off mouse is its only works on the male mice. To address these limitations, we generated and characterized a new Myh11-CreER T2 mouse ( Myh11-CreER T2 -KI ), which results from integration of a CreER T2 cassette into the endogenous Myh11 gene locus, enabling faithful SMC-specific lineage tracing or specific gene knockout in SMC in vivo in both sexes. Method: A tamoxifen-inducible, CreER T2 cassette was inserted into one of the Myh11 gene alleles after the initiation codon of Myh11 . To validate the Myh11 CreERT2/+ -mediated gene recombination in SMCs, Myh11-CreER T2 -KI mice were crossed with mTmG dual reporter mice or the lncRNA Carmn GFP knock-in reporter/knockout mice to trace SMC lineages or generate SMC-specific inducible Carmn knockout mice. The recombination specificity in SMCs was analyzed by GFP fluorescence analysis. Results: The CreER T2 cassette inserted into the Myh11 gene locus negligibly affects endogenous MYH11 protein expression. In mTmG / Myh11-CreER T2 -KI reporter mice, GFP expression was detected specifically in both vascular and visceral SMCs in various tissues after tamoxifen mediated Cre activation. Moreover, Myh11-CreER T2 -KI -mediated Carmn SMC-specific deletion in adult female mice recapitulated the lethal intestinal pseudo-obstructive phenotype observed in male Myh11-CreER T2 -Off Carmn KO mice. Conclusions: Our new Myh11-CreER T2 -KI mouse model provides an additional genetic tool using the fidelity of an endogenous SMC gene locus to trace SMC lineages and delete genes in the SMC of both sexes of mice.