Villin is an actin-binding protein that is encoded by Vil1. The transgenic mice in which Vil1 acts as a promoter of Cre are widely used to inactivate the intestinal epithelium’s genes. These lines have been well-characterized in the intestine; however, their extraintestinal Cre activity has not been entirely explained or analyzed. The Tg(Vil1-cre)1000Gum and Tg(Vil1-cre)997Gum transgenic lines were studied, in which a 12.4-kilobase region of the Vil1 promoter drives Cre expression. These were then crossed with Rosa26-Ai9 reporter mice, in which Cre-mediated excision of a stop cassette leads to the expression of fluorescent TdTomato.

There was no GFP expression in oviduct or ovary, but germ cell expression was observed within a subset of seminiferous tubules in the testis in all males, raising the possibility of germline recombination. The Vil1CreERT2/23 line is unique among the four lines studied because it expresses a Cre recombinase–estrogen receptor fusion protein that requires tamoxifen to trigger nuclear translocation and recombination. Notably, both Vil1CreERT2/23 and Vil1Cre/20 lines exhibited Cre activity in a subset of male germ cells, raising concern that when these lines are used to delete genes, some progeny could have globally null alleles. One limitation of our study is that two different Cre-reporter lines were used, although both targeted the Rosa26 locus. Recombination efficiency at different loxP sites and genomic loci can vary.

Thus, the findings guide Cre activity in these four Vil1 transgenic lines and this activity may manifest differently in the context of other floxed alleles. The data gathered shows that Cre lines commonly used to study intestinal epithelial biology can be active in the nervous system and other issues that might affect gut functions. These observations do not detract from these genetic tools’ value, but they emphasize the need for careful reagent choice, data interpretation, and methods reporting in all studies using these mice.