A New Autosomal Myh11-CreERT2 Smooth Muscle Cell Lineage Tracing and Gene Knockout Mouse Model-Brief Report

Abstract

Background: The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreER^T2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreER^T2 mouse (referred to as Myh11-CreER^T2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice.

Methods: A Myh11-CreER^T2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ER^T2 after the Myh11 start codon. Myh11-CreER^T2-RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses.

Results: Myh11-CreER^T2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreER^T2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreER^T2 mice. Labeling was equivalent in both male and female Cre⁺ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence.

Conclusions: We generated and validated the function of an autosomal Myh11-CreER^T2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

Keywords: arteries; female; mice, transgenic; myocytes, smooth muscle; myosin heavy chains; tamoxifen.

Figure 1:. Myh11-CreER^T2-RAD efficiently induces floxed tdTomato reporter gene recombination and subsequent lineage tracing of SMC in the BCA and Aorta analogous to Myh11-CreER^T2.

A.) Schematic showing the experimental design of tamoxifen-induced recombination of floxed fluorescent reporter genes in Myh11-CreER^T2-RAD mice. B.) Schematic showing the experimental design to determine fluorescent reporter gene expression in the BCA and intact aorta driven by Myh11-CreER^T2-RAD using immunofluorescence imaging and flow cytometry respectively. C.) Representative images showing tamoxifen-induced tdTomato expression in SMCs of the BCA in Cre⁺ Myh11-CreER^T2-Off (left) and Myh11-CreER^T2-RAD (right) mice. D.) Quantification of % tdTomato⁺/ACTA2⁺ cells in the BCAs of Cre⁺ Myh11-CreER^T2-Off (n=5) and Myh11-CreER^T2-RAD (n=7) from immunofluorescence images. E.) Zoomed in Myh11-CreER^T2-RAD BCA image showing tdTomato expression (red) in the medial SMCs between the EEL and IEL (dashed lines) but not in the EC layer (arrow) or adventitia (arrowhead), L=lumen. F.) Quantification of flow cytometry results of aortic cells from Myh11-CreER^T2-Off (n=5 Cre⁻ females, n=5 Cre⁺ males) and Myh11-CreER^T2-RAD (n=8 Cre⁻, n=8 Cre⁺, both sexes) mice fed tamoxifen. Scale Bar=100μm. Schematics created with BioRender.com

Figure 2:. Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD can induce tamoxifen independent recombination of floxed fluorescent reporter genes which is locus-dependent.

Mice were treated as shown in Figure 1B except mice were fed standard rodent diet (TMX−) at 6–8 weeks in lieu of tamoxifen diet (TMX+). A.) Representative immunofluorescent images of BCAs from Myh11-CreER^T2-Off/tdTom (left) and Myh11-CreER^T2-RAD/tdTom (right) showing tamoxifen-independent tdTomato expression in Cre⁺ (but not Cre⁻) mice. B.) Representative immunofluorescent images of BCAs from Myh11-CreER^T2-Off/eYFP (left) and Myh11-CreER^T2-RAD/eYFP (right) showing no detectable tamoxifen-independent eYFP expression in either Cre⁺ or Cre⁻ mice. C.) Quantification of flow cytometry results of cells dissociated from intact aortas from standard rodent diet fed (TMX−) Myh11-CreER^T2-Off/tdTom (n=5 Cre⁻ females, n=5 Cre⁺ males) and Myh11-CreER^T2-RAD/tdTom mice (n=8 Cre⁻, n=8 Cre⁺ both sexes). D.) Quantification of flow cytometry results of cells dissociated from intact aortas from standard rodent diet fed (−TMX) Myh11-CreER^T2-Off/eYFP (n=3 Cre⁻ females, n=5 Cre⁺ males) and Myh11-CreER^T2-RAD/eYFP mice (n=7 Cre⁻, n=6 Cre⁺ both sexes). E.) and F.) Schematics depicting the relative PCR recombination products generated after LoxP excision (black arrowheads) for tdTomato and eYFP reporter genes respectively. G.) PCR recombination analysis of the tdTomato reporter in Myh11-CreER^T2-Off/tdTom (top) and Myh11-CreER^T2-RAD/tdTom mice (bottom). H.) PCR recombination analysis of the eYFP reporter in Myh11-CreER^T2-Off/eYFP (top) and Myh11-CreER^T2-RAD/eYFP mice (bottom). Schematics created with BioRender.com.