Mlc2v-Cre induces germline recombination in a sex-biased manner

The myosin light chain-2 ventricular isoform (MLC2V), encoded by the Myl2 gene, is a sarcomeric protein in ventricular cardiomyocytes, making Mlc2v-Cre mice (Myl2^{tm1(cre)Krc/AchakJ}) a valuable tool for ventricle-specific gene targeting. However, we observed unexpected recombination in nonmuscular tissues of offspring from Mlc2v-Cre breeders, suggesting off-target Cre activity and germline transmission. This study aims to quantify the prevalence and sex-dependency of off-target recombination in Mlc2v-Cre mice. To address this, Mlc2v-Cre mice were crossed with Rosa26^tdTomato reporter mice, and Cre-Lox recombination was visualized in the resulting embryos. Our results demonstrate that Mlc2v-Cre induces germline recombination in both male and female breeders, with a higher incidence in females, leading to the unintended generation of a whole body recombined allele, independent of Cre transgene, in the offspring. This was further supported by MLC2V and tdTomato expression in male and female germ cells. These findings highlight the importance of validating Cre-mediated recombination specificity to avoid confounding experiment outcomes and ensure accurate data interpretation.NEW & NOTEWORTHY Although the Mlc2v-Cre mouse line is widely used for ventricle-specific gene targeting in cardiac research, this study shows that both male and female Mlc2v-Cre mice can cause unexpected germline recombination, with a higher incidence in females. To avoid confounding results from whole body gene targeting, these findings underscore the need to validate off-target recombination.