Impaired skeletal muscle regeneration induced by Cre recombinase activation in skeletal muscle stem cells

The value of the Cre-lox system in biology is well-recognized, which is reflected by its widespread use to assess the role of a gene in a specific tissue or cell-type. Not in the least, Cre recombinase expressed under the Pax7 promotor has been invaluable for the study of skeletal muscle stem cell (MuSC) biology. In this study, we aimed to systematically assess the effects of the genetic makeup of Pax7Cre mice and Tamoxifen (Tx) treatment on skeletal muscle regeneration. We demonstrate that Tx treatment per se does not affect skeletal muscle regeneration at 14 days post injury (d.p.i.) induced by cardiotoxin, but specifically worsened regeneration in two Pax7CreERT2 lines. Pax7 heterozygosity in Pax7CreERT2(FAN) mice resulted in a lower body mass and Tibialis Anterior (TA) mass, a higher number of fibers per section, and a lower number of Pax7+ cells than in Pax7CreERT2(GAKA) mice, but Tx treatment did not worsen these effects caused by Pax7 haploinsufficiency. In vitro, proliferation of Pax7CreERT2(FAN) MuSCs was impaired after 4-Hydrotamoxifen (4-OHT) treatment, while cell survival and differentiation remained unaffected. Together with a lower number of nuclei per fiber after Tx treatment in Pax7CreERT2(FAN) and male Pax7CreERT2(GAKA) mice, this may suggest an impaired MuSC pool expansion upon Cre activation. Yet, the in vivo MuSC pool was maintained in Pax7Cre mice at 14 d.p.i. Overall, our results directly show that Cre recombinase activity has an off-target effect on MuSCs, which warrants the use of Tx-treated Pax7CreERT2 mice as experimental controls in future studies, and demand caution in interpreting data using other controls in previous studies.