To determine whether inversion of Psg22 relative to flanking Psg genes is a recent event confined to a limited number of laboratory strains, or an older variant that may predate divergence of major mouse strains and indeed species, we surveyed the Mouse Genomes Project. Psg locus genomic sequences of 17 mouse strains were analysed by sequence similarity searches, genomic location, and alignment. WefoundPsg22 inversion in 16 of 17 mouse strains analysed (129SI_SvlmJ, AJ, AKR_J, Balb_cJ, C3H_HeJ, C57BL_6NJ, Cast_EiJ, CBA_J, DBA_2J, FVB_NJ, LP_J, NOD_ShiLtJ, NZO_HlLtJ, PWK_PhJ, Spretus_EiJ, WSB_EiJ), including the most early diverging M. spretus;the Psg22 transcript could not be clearly identified in the M. caroli/EiJ strain due to poor sequence quality. This phylogenetic distribution most likely indicates an inversion of Psg22 at least 1.7 million years ago that has been maintained in the mouse clade. Therefore, this inversion may underpin a placenta phenotype or influence fetal growth or survival.
We have produced Psg22-null mutant mouse strains by pronuclear microinjection of fertilised oocytes with a CRISPR-Cas9 pX458 vector targeting a site in the Psg22 open reading frame (ORF) in the exon 2 (Figure A), which was previously tested for activity in the NIH-3T3 embryonic fibroblast cell line (data not shown). Seventy-two microinjected B6D2F2 zygotes were transferred to oviducts of three CD1 pseudopregnant female recipients, and 25 offsprings were ear-clipped for identification and DNA extraction. PCR genotyping was performed using Psg22 gene-specific primers flanking the CRISPR target site, followed by Cel I assay and direct sequencing of Cel I assay-positive PCR products. Eight progeny had mutations, and BLAST alignment indicated that five were genetic mosaics, two had deletions of 10 and 16 bp, respectively, and one had a 16-bp deletion just 3′ of a 1-bp substitution.
We selected two mutant founders for breeding: one with a 10-bp deletion (Psg22Δ10) and one exhibiting a 16-bp deletion 3' of a 1-bp substitution (Psg22Δ16) (Figure B). Both mutations are predicted to cause a frameshift resulting in a premature stop codon and were associated with greatly reduced mRNA levels (Figure C), possibly due to nonsense-mediated decay.
Both founders were backcrossed to the C57BL/6 strain for two generations (to give an average of 87.5% C57BL/6 background) before intercrossing heterozygous male and female littermates of the Psg22Δ10 and Psg22Δ16 strains to determine genotype transmission ratios. Combining the data from the two Psg22-mutant strains, there were 115 offsprings: 28 homozygous wildtype, 30 homozygous null, and 57 heterozygotes, consistent with Mendelian expectations, and suggesting that Psg22 is not essential for the development (individual strain data and the statistics are given in Figure D). Subsequent breeding of the Psg22Δ10/Δ10 and Psg22Δ16/Δ16 homozygotes confirmed that these genotypes are fertile (data not shown). This suggests that Psg22 is dispensable for fertility and reproduction under laboratory conditions.
Previous reports indicate that reproductive phenotypes may be elicited by applying stressors such as hypoxia to pregnancy in mouse strains with the mutations of placenta-expressed genes. We intercrossed heterozygous male and female littermates of the Psg22Δ10 and Psg22Δ16 strains as described above and, using environmental chambers, applied hypoxic stress (11% O2) to pregnant females for either 5 days (E5-E10) or 10 days (E5-E15). At E17 or E18, pregnant females were sacrificed and embryos were genotyped and corresponding placentas were fixed, weighed, and analysed using stereological techniques. Similar to unstressed pregnancies, there was no deviation from expected Mendelian ratios (Figure D), and there was no difference in placental weight and anatomy among the genotypes (Figure E and F), indicating that Psg22 expression is dispensable for successful pregnancy under hypoxic conditions.