An Inducible Cre Mouse with Preferential Activity in Vascular Smooth Muscle Evades a Previously Lethal Intestinal Phenotype

Abstract

All smooth muscle cell (SMC) restricted Cre mice recombine floxed alleles in vascular and visceral SMCs. We generated a new tamoxifen-inducible CreERT2 mouse, Itga8-CreERT2, and compared its activity to the widely used Myh11-CreERT2 mouse. Both CreERT2 mice showed similar activity in vascular SMCs; however, Itga8-CreERT2 displayed limited activity in visceral SMC-containing tissues (e.g., intestine). Myh11-CreERT2 (but not Itga8-CreERT2) mice displayed high levels of CreERT2 protein, tamoxifen-independent activity, and an altered transcriptome. Whereas Myh11-CreERT2-mediated knockout of Srf resulted in a lethal intestinal phenotype, loss of Srf with Itga8-CreERT2 (SrfItga8) revealed viable mice with attenuated vascular SMC contractile gene expression, but no evidence of intestinal pathology. Male and female SrfItga8 mice presented with vascular contractile incompetence; however, only male SrfItga8 mice showed systemic changes in blood pressure. These results establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre strains, including Myh11-CreERT2, where SMC gene loss results in visceral myopathies that obfuscate accurate phenotyping in vascular SMCs.

A vascular smooth muscle-specific integrin-α8 Cre mouse for lymphatic contraction studies that allows male-female comparisons and avoids visceral myopathy

“…Taken together with the expression data in Figure 2, these observations also suggest that submaximal recombination of the Rosa26mTmG reporter driven by either of the two different Cre lines tested does not preclude effective deletion of Cav1.2 in lymphatic SM. A similar conclusion regarding deletion of the Srf gene was reached in the study by Miano and colleagues (Warthi et al, 2022), and locus-specific or flox-site disparities in recombination efficiency with inducible Cre lines has been widely reported (Stifter and Greter, 2020). Critically, as with Myh11-CreER T2 ;Cav1.2 f/f mice, Itga8-CreER T2 ; Cav1.2 f/f mice did not exhibit any signs of peripheral edema (e.g., footpad swelling) even in the complete absence of active lymphatic contractions.…”

“…Myh11-CreER T2 mice [B6.FVB-Tg (Myh11-cre/ERT2)1Soff/J] were originally obtained from Stefan Offermanns (Max-Planck Institute, Bad Neuheim). Itga8-CreER T2 mice were obtained from Joe Miano and Lin Gan (Medical College of Georgia at Augusta University [Warthi et al, 2022)]. Both strains were bred with Ca v 1.2 f/f mice (Cacna1c tm3Hfm /J; JAX #024714) or Rosa26mT/mG f/f reporter mice (JAX, #007676), to generate Myh11-CreER T2 ;Ca v 1.2 f/f , Itga8-CreER T2 ;Ca v 1.2 f/f , Myh11-CreER T2 ;Rosa26mT/mG f/f , and Itga8-CreER T2 ;Rosa26mT/mG f/f mice.…”

“…We recently reported the efficiency of recombination for the Rosa26 mT/mG reporter in lymphatics from Myh11-CreER T2 ; Rosa26mT/mG f/f vs. Itga8-CreER T2 ;Rosa26mT/mG f/f mice [Figure 3 in ref (Warthi et al, 2022)]. Here, we reanalyzed a subset of that data (2 male Itga8-CreER T2 ;Rosa26mT/mG f/f mice) along with data from two (new) female Itga8-CreER T2 ;Rosa26mT/ mG f/f mice to compare the recombination efficiency in lymphatics from mice of different sexes.…”

“…Here, we tested the effectiveness of a new vascular SM-specific Cre, based on the integrin α8 promoter (Itga8-CreER T2 ), recently developed and characterized by Miano and colleagues (Warthi et al, 2022), to assess the effects of Cav1.2 deletion on lymphatic function. This Cre was demonstrated to be much more specific for vascular than visceral SM based on multiple indices, including the important advantage that deletion of genes such as Srf (Itga8-CreER T2 ;Srf f/f mice) avoided an otherwise lethal gastrointestinal phenotype (Warthi et al, 2022). Itga8-CreER T2 can be used to delete genes in both male and female mice, potentially allowing studies of sex differences.…”

“…Recombination in lymphatic smooth muscle was shown to be somewhat less efficient than with the Myh11-based Cre, based on GFP expression, as lymphatic vessels from Itga8-CreER T2 ;Rosa26mTmG f/f mice expressed GFP in 65% of lymphatic SM cells compared to 90% of lymphatic SM cells in Myh11-CreER T2 ;Rosa26mTmG f/f mice [Figure 3 in (Warthi et al, 2022)]. However, recombination efficiencies based on the excision of stop floxed cassettes in the mTmG reporter and an inducible Myocardin transgene as well as a floxed Srf mouse (Miano et al, 2004) were more comparable to those when Myh11-CreER T2 was used (Warthi et al, 2022). The goal of the present study was to use Itga8-CreER T2 to delete Cav1.2 and assess the impact on mouse survival and lymphatic contractile function.…”