Generation of a novel Stra8-driven Cre recombinase strain for use in pre-meiotic germ cells in mice†

Abstract

The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility.

Keywords: Cre recombinase; Stra8; germ cell; reporter mouse line.

Graphical abstract

Figure 1

Schematic representation for generation of the Stra8^P2Acre knockin allele. (A) The endogenous Stra8 transcript (top) showing location of the homology arms for CRISPR/Cas9 targeting. The Stra8^P2Acre allele produces a bicistronic transcript that includes a self-cleaving P2A site and cre recombinase expression cassette in frame to exon 10 of Stra8, keeping the endogenous coding sequence and 5′ and 3' UTR intact. (B) Genotyping PCR for offspring from a Stra8^P2Acre/+ × Stra8^P2Acre/+ using primers annotated above. Agarose gel of amplification products from genomic DNA PCR using insert-specific primers distinguish between wild-type (WT), heterozygous (KI/+), or homozygous knockin (KI/KI). The H2O lane is the negative control without genomic DNA.

Figure 2

Stra8^P2Acre/+ is active in germ cells in E13.5 ovary. (A) Whole mount ovary immunofluorescence using an antibody against tdTomato (tdTom; red) indicated recombination by Cre in anteromedial ovary (OV), but not the mesonephros (ME), in Stra8^P2Acre/+ tdTomato + (A–H) compared to cre negative littermates (I–L). (B, F, J) Tissue was co-stained with an antibody against the germ cell marker, TRA98 (green) and counterstained with DAPI (C, G, K). (D, H, L) Merged image of tdTomato, TRA98, and DAPI. Panels E–H show zoomed-in images of ovary region boxed in panel D. Images were taken on an LSM 780 confocal microscope. Anatomic position is shown by the cross in panel D, with A, anterior; P, posterior; L, lateral; M, medial. Scale bar = A–D, 100 μm; E-L, 20 μm.

Figure 3

Stra8^P2Acre/+ shows widespread recombination in germ cells in the E15.5 ovary. Widespread activity of Stra8-driven cre recombinase shown in tdTomato reporter mouse ovaries (Stra8^P2Acre/+ tdTomato+) (A–D) but not control ovaries (Stra8 ^+/+ tdTomato+) (E–H) at E15.5. Red fluorescence (tdTom) indicates activity of Stra8^P2Acre/+. Whole mount ovaries were co-stained with the germ cell marker TRA98 (green) (B, F) and counterstained with DAPI (gray; C, G). Merged images are shown in D, H. Images were taken on an LSM 780 Confocal Microscope. Scale bar = 20 μm.

Figure 4

Widespread recombination in oocytes of Stra8^P2Acre/+ tdTomato+ ovary at PND0. (A–D) Example of immunofluorescent imaging of Stra8^P2Acre/+ tdTomato+ (Cre Pos) and control ovaries (tdTomato+ Cre Neg) (E–H) at PND0 ovary for tdTomato (A) the germ cell marker, TRA98 (B), and counterstained with DAPI (C). Red fluorescence (tdTomato) (A, E) indicates recombination. Immunofluorescent co-staining was performed on ovary cryosections, and images taken on an LSM 780 confocal microscope. Exposure times were the same for both cre negative and cre positive sections. Scale bar = A–C, E–G, 50 μm; D, H, 20 μm.

Figure 5

Recombination of tdTomato + is detectable at PND3 in Stra8^P2Acre/+ tdTomato + testes in germ cells. (A) tdTomato immunofluorescence in PND3 Stra8^P2Acre/+ tdTomato + testis cryosections co-stained for TRA98 to mark germ cells (B) and DAPI (C) with merged images shown in panel D. (E) tdTomato immunofluorescence in PND3 wild type (cre neg) littermate cryosections with TRA98 (F), DAPI (G) and merged images (H) shown for comparison. Red fluorescence indicates recombination by the enzymatic actions of Stra8-cre, thus showing endogenous Stra8 expression. Germ cells are marked with the anti-TRA98 antibody. Images were taken on an LSM 780 confocal microscope. Scale bar = 20 μm.

Figure 6

Recombination of tdTomato + is detectable at PND5 in Stra8^P2Acre/+ testes. (A) Endogenous tdTomato fluorescence in PND5 Stra8^P2Acre/+ tdTomato + testis cryosections, co-stained with (B) TRA98 to mark germ cells and (C) DAPI with merged images shown in panel D. Endogenous tdTomato fluorescence in PND5 wild type (cre neg) littermate cryosections shown in panel E with (F) TRA98, (G) DAPI and (H) merged images shown for comparison. Red fluorescence indicates recombination by the enzymatic actions of Stra8-cre, thus showing endogenous Stra8 expression. Images were taken on an LSM 780 confocal microscope. Scale bar = 50 μm.

Figure 7

Stra8^P2Acre mice have normal fertility. (A) Average pups per litter and (B) average litters per month calculated for n = 3–4 breeding animals. Shown are data for wild type (WT) cre negative, heterozygous Stra8^P2Acre/+ (KI/+) and homozygous Stra8^{P2Acre/P2Acre} (KI/KI) littermates bred to WT. Both graphs shown as mean + s.e.m. Error bars are not visible on control male litters per month bars due to values of 1 ± 0. Analysis by one-way ANOVA with multiple comparisons indicated no significant difference (ns) between genotypes.