A New Autosomal Myh11-CreER ^T2 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 T 2 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 T 2 mouse (referred to as Myh11-CreER T 2 -RAD ), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. Methods: A Myh11-CreER T 2 -RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ER T 2 after the Myh11 start codon. Myh11-CreER T 2 -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 T 2 -RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreER T 2 -RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreER T 2 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 T 2 -RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

“Cre”ating New Tools for Smooth Muscle Analysis

“…In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Deaton et al 17 describe the generation of a novel bacterial artificial chromosome–based Myh11-CreER T2 -RAD mouse line, in which bacterial artificial chromosome insertion occurred on chromosome 2. The authors present a rigorous side-by-side analysis of the old and the new Myh11-CreER T2 mouse lines and provide robust data indicating that the new line is equivalent to the original with the advantage of allowing experiments in male and in female Myh11-CreER T2 mice (Figure 1).…”

“…This leakiness is SMC-specific and is also seen in the original Myh11-CreER T2 -Off line. 17 It is most likely due to a small fraction of the CreER T 2 protein which escapes the cytoplasmic sequestration mechanisms in the absence of tamoxifen, including binding to heat shock protein 90, and then translocates to the nucleus. Interestingly, Deaton et al 17 demonstrated that the degree of age-dependent tamoxifen-independent recombination varies between different floxed alleles.…”

“…17 It is most likely due to a small fraction of the CreER T 2 protein which escapes the cytoplasmic sequestration mechanisms in the absence of tamoxifen, including binding to heat shock protein 90, and then translocates to the nucleus. Interestingly, Deaton et al 17 demonstrated that the degree of age-dependent tamoxifen-independent recombination varies between different floxed alleles. Thus, tamoxifen-independent recombination, which has been observed in many CreER T 2 -driver mice, 19 requires appropriate controls, especially when working with older mice.…”

“…Prior work from the Owens’ lab highlights another potential confounder in that the peanut oil vehicle was shown to cause autofluorescence in adipose-tissue macrophages, 22 which could lead to mistaken identification of some macrophages as SMC-derived. Deaton et al 17 circumvented this challenge by oral tamoxifen administration. In cases where shorter-term tamoxifen delivery is required, as in developmental studies, this caveat must be considered.…”

Commonly Used Myh11-CreER T2 Strain Carries a Y-Linked Functional Wild-Type Tlr7 Allele

“…4 These facts together with our findings demand further studies to fully characterize the genetic defects and their biological consequences for the Myh11-CreER mouse, so that critical information may be applied when revisiting previous studies involving the use of this mouse. The newly developed autosomal Myh11-CreER 5 and Itga8-CreER 3 mouse lines may serve well for this purpose.…”