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

The myosin light chain-2 ventricular isoform (MLC2V), encoded by the <i>Myl2</i> gene, is a sarcomeric protein in ventricular cardiomyocytes, making Mlc2v-Cre mice (<i>Myl2</i>^{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 <i>Rosa26</i>^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 <i>Cre</i> 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.<b>NEW & NOTEWORTHY</b> 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.