iCre recombinase expressed in the anti-Müllerian hormone receptor 2 gene causes global genetic modification in the...

Genetically engineered mice are widely used to study the impact of altered gene expression in vivo. Within the reproductive tract, the Amhr2-IRES-Cre(Bhr) mouse model is used to ablate genes in ovarian granulosa and uterine stromal cells. There are reports of Amhr2-IRES-Cre(Bhr) inducing recombination in non-target tissues. We hypothesized the inefficiency or off-target Cre action in Amhr2-IRES-Cre(Bhr) mice is due to lack of recombination in every cell that expresses Amhr2. To investigate, we created a new targeted knock-in mouse model, Amhr2-iCre(Fjd), by inserting a codon-optimized improved Cre (iCre) into exon 1 of the Amhr2 gene. Amhr2-iCre(Fjd)/+ males were mated with females that contain a lox-stop-lox cassette in the Sun1 gene so when DNA recombination occurs, SUN1-sfGFP fusion protein is expressed in a peri-nuclear pattern. In adult Amhr2-iCre(Fjd)/+ Sun1LsL/+ mice, Amhr2-iCre(Fjd)-mediated genetic recombination was apparent in uterine epithelial, stromal, and myometrial cells, while Amhr2-IRES-Cre(Bhr)/+ Sun1LsL/+ females demonstrated inter-mouse variability of Amhr2-IRES-Cre(Bhr) activity in uterine cells. Fluorescence was observed in Amhr2-iCre(Fjd)-positive mice at post-natal Day 1, indicating global genetic recombination, while fluorescence of individual Amhr2-IRES-Cre(Bhr)-positive pups varied. To determine the developmental stage that genetic recombination first occurs, Sun1LsL/LsL females were super-ovulated and mated with Amhr2-IRES-Cre(Bhr)/+ or Amhr2(iCre/+)Fjd males, then putative zygotes were collected and cultured. In the four-cell embryo, Amhr2-iCre(Fjd) and Amhr2-IRES-Cre(Bhr) activities were apparent in 100% and 25-100% of cells, respectively. In conclusion, Amhr2-IRES-Cre(Bhr) or Amhr2-iCre(Fjd) driven by the Amhr2 promoter is active in the early embryo and can lead to global genetic modification, rendering this transgenic mouse model ineffective.