A shortened tamoxifen induction scheme to induce CreER recombinase without side effects on the male mouse skeleton

•The off-target action of tamoxifen represents a pitfall to avoid in CreER technology.•Shortening tamoxifen induction regimens could avoid the unintended effects.•Androgen-sensitive tissues including bone were preserved only by a shortened protocol.•Cre-mediated gene inactivation was not boosted by switching to a standard protocol.•We validated a novel tamoxifen induction regimen without off-target effects on bone.

The selective estrogen receptor modulator tamoxifen exerts estrogen agonistic or antagonistic actions on several tissues, including bone. The off-target effects of tamoxifen are one of the most widely recognized pitfalls of tamoxifen-inducible Cre recombinases (CreERs), potentially confounding the phenotypic findings. Still, the validation of tamoxifen induction schemes that minimize the side effects of the drug has not been addressed. Here, we compared the side effects on the skeleton and other androgen-responsive targets of a shortened tamoxifen regimen (2 doses of 190 mg/kg body weight by oral gavage) to a standard protocol (4 doses) and determined their efficiency in inducing CreER-mediated gene deletion. In addition, both a vehicle- and a 10-dose group, which served as a positive control for tamoxifen side effects, were also included. For this purpose, we generated male mice with a floxed androgen receptor (AR) and a neuron-specifically expressed CreER. Treatment with two doses of tamoxifen was the only regimen that did not diminish androgenic bioactivity, as assessed by both seminal vesicles and levator ani/bulbocavernosus muscle weights and serum testosterone concentrations. Similarly, trabecular and cortical femoral bone structure were dramatically altered by both the standard and high-dose protocols but not by the shortened version. Serum osteocalcin and bone-gene expression analyses confirmed the absence of effects on bone by 2 doses of tamoxifen. This protocol decreased AR mRNA levels efficiently and specifically in the nervous system. Thus, we optimized a protocol for tamoxifen-induced CreER gene deletion in mice without off-target effects on bone and male reproductive organs.