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

Abstract

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.

Keywords: genetics; global genetic modification; transgenic mice.

Graphical abstract

Figure 1

Targeting strategy to make the Amhr2-iCre(Fjd) allele. CRISPR/Cas9-mediated targeting strategy to generate the Amhr2-iCre(Fjd) allele. The iCre ORF and polyA signal were inserted immediately downstream of the start codon in exon 1. Locus-specific PCR screens and Transnetyx primer/probe genotyping assays are indicated in the final allele.

Figure 2

Amhr2-iCre(Fjd) activity in the adult uterus. Adult female mice (n = 6) aged 7 to 12 weeks were euthanized and the uterus was assessed for Amhr2-iCre(Fjd) activity and SUN1-sfGFP fusion protein expression. A cross-section of the whole uterus from an Amhr2-iCre(Fjd)/+ Sun1^LsL/+ mouse depicts SUN1-sfGFP expression throughout the tissue (A). Close-ups of the uterus show SUN1-sfGFP expression indicative of Amhr2-iCre(Fjd) activity in epithelial, stromal, and myometrial cells (B, C). Wild-type (Cre negative) mice did not express GFP in the uterus (D–F). Blue indicates nuclear staining and green is GFP.

Figure 3

Amhr2-iCre(Fjd) activity in neonatal pups. Pups from Amhr2-iCre(Fjd)/+ mice crossed with Sun1^LsL/LsL mice were assessed the day after being born (post-natal day 1; (n = 24; 3 litters). Pups that were Amhr2-iCre(Fjd)/+ positive appeared fluorescent indicative of SUN1-sfGFP expression and Amhr2-iCre(Fjd) activity (A–C). Wild-type (WT; Cre negative) mice did not appear green (D).

Figure 4

Amhr2-IRES-Cre(Bhr) activity is variable in adult tissues and neonatal pups. Adult female mice (n = 6) aged 7–12 weeks were euthanized and the uterus was assessed for Amhr2-IRES-Cre(Bhr) activity and SUN1-sfGFP expression. Uteri from Amhr2-IRES-Cre(Bhr)/+ Sun1^LsL/+ mice had variation in the expression of SUN1-sfGFP fusion protein with some mice lacking epithelial recombination (A–C) and some mice having strong recombination in epithelial, stromal, and myometrial cells (D–F). Blue indicates nuclear staining and green is GFP. To assess Amhr2-IRES-Cre(Bhr) activity in neonatal mice, pups (n = 28; 3 litters) were collected at post-natal day 1 from Amhr2-IRES-Cre(Bhr)/+ male mice mated with Sun1^LsLLsL+ females. There was wide variation of fluorescence in Amhr2-IRES-Cre(Bhr)/+ pups (G). Wild-type (Cre negative) mice did not appear green (H).

Figure 5

Embryo culture depicts Amhr2-iCre(Fjd) and Amhr2-IRES-Cre(Bhr) activity in the early embryo. Female Sun1^LsL/Lsl mice were super-ovulated and mated with either Amhr2-IRES-Cre(Bhr)/+ or Amhr2-iCre(Fjd)/+ male mice. The morning after mating, female mice were euthanized, and putative zygotes were recovered and placed in culture. SUN1-sfGFP expression is apparent as early as the two- to four-cell stage in some embryos, indicating Amhr2-IRES-Cre(Bhr) and Amhr2-iCre(Fjd) activity. By the blastocyst stage, the majority of cells are SUN1-sfGFP positive, indicating Amhr2-IRES-Cre(Bhr) or Amhr2-iCre(Fjd) recombination has occurred. Genetic recombination this early in development demonstrates that Amhr2-IRES-Cre(Bhr) and Amhr2-iCre(Fjd) are not specific to the reproductive tract. Blue is a nuclear stain (Hoechst 33342), and green is the endogenous expression of sfGFP.