A dataset of 17 mouse strains was assembled from the Sanger Institute website (ftp://ftp-mouse.sanger.ac.uk/REL-1504-Assembly), as follows: 129SI_SvlmJ, AJ, AKR_J, Balb_cJ, C3H_HeJ, C57BL_6NJ, Cast_EiJ, Caroli_EiJ, CBA_J, DBA_2J, FVB_NJ, LP_J, NOD_ShiLtJ, NZO_HlLtJ, PWK_PhJ, Spretus_EiJ, WSB_EiJ. The region known to contain the Psg cluster, i.e. region 15,000,000–22,000,000 on chromosome 7, was extracted from all mouse strains from the whole-genome file using a custom Bash script. Psg gene sequences were obtained from Ensembl for the mouse reference strain C57BL/6J, and these were used as the query sequences in the subsequent sequence similarity searches. Nucleotide BLAST was used to obtain Psg gene sequences from all mouse strains using the annotated Psg sequences from C57BL/6J. Coordinates from the BLAST search allowed localisation of the queried Psg genes on either leading or lagging strand in the different strains.
CRIPSR-Cas9 vector cloning and validation
Psg22 targeting oligonucleotides were selected using CHOPCHOP (https://chopchop.rc.fas.harvard.edu) and were chosen from exon 2 of the Psg22 to disrupt the ORF. Oligonucleotides (Eurofins MWG Operon, Eberberg, Germany) were cloned into px458 CRISPR vectors (www.addgene.org) using Zhang laboratory protocol (www.genome-engineering.org/crispr/). The px458-Psg22 vector was tested in the NIH-3T3 cell line using transient transfection with 3 µL Turbofect transfection reagent. 72 h post-transfection, cells were harvested and genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue kit. 10 ng DNA was used as template for PCR reaction using Psg22 specific primers spanning the CRISPR target site; the resulting PCR products were analysed for successful genome editing using Surveyor nuclease Cel I assay kit (Integrated DNA Technologies, www.idtdna.com).
Psg22 -mutant mouse production
Male parental B6D2F1 mice and female B6D2F1 zygote donor mice were obtained from DBA/2 and C57BL/6J breeding pairs. B6D2F1 females were superovulated at 6–10 weeks of age by intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG) 48 h apart. Directly after hCG injection, the superovulated females were mated to B6D2F1 males. CD1 pseudopregnant females were set up in the afternoon of day 1 by mating with vasectomised CD1 males. The following morning (day 0.5 postcoitum; embryonic day 1, E1), B6D2F1 zygotes were harvested and placed in M16 drops in a humidified 5% CO2 incubator at 37°C. The vector px458-Psg22 was diluted to 5 ng/µL in microinjection buffer (8 mM Tris-HCl, 0.15 mM EDTA). Pronuclear microinjection was carried out in a drop of M2 medium under mineral oil. The same day, 72 microinjected zygotes were surgically transferred into the oviducts of 3 CD1 pseudopregnant females. Each CD1 female was bilaterally transferred with 24 microinjected zygotes. 25 weaned offsprings were ear-clipped and genotyped. Genotyping primers were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify a 606-bp Psg22- specific product spanning the CRISPR-Cas9 target site. Genotyping primers were Psg22-2F 5′-CAGAAACAACACCATGTGAGT-3′ and Psg22-2R 5′-AGACACAATGCAGAGGGAAATA-3′. PCR products were purified (QIAGEN PCR purification kit) and directly sequenced (GATC, Koln, Germany).
Hypoxia treatment of pregnant mice
Commencing at E5 of pregnancy, female mice were housed in standard cages placed in commercial environmental chambers with precise control of ambient oxygen concentration (Oxycycler, Biospherix, NY) which was maintained at 11% balance nitrogen for 5 or 10 consecutive days (until E10 or E15, respectively). On removal from hypoxia, mice were housed under normal husbandry conditions in room air until E17 or E18.
Quantitative reverse transcription polymerase chain reaction
First-strand cDNA was synthesised using 1 µg total RNA in a 20 µL reaction using random hexamer priming and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, UK). Quantitative RT-PCR (qRT-PCR) primers were designed to give unbiased amplification of Psg22 transcripts: Psg22-QRT-F 5′-CGCATGGCCAGTTGGCCATT-3′ and Psg22-QRT-R 5′-AAAGCGGGGGAAATAGTTGTAGTA-3′. Hypoxanthine guanine phosphoribosyltransferase (Hprt) expression was used to normalise mRNA input and cDNA synthesis efficiency using primers: Hprt-F: 5’-CTATAAGTTCTTTGCTGACCTGCT-3’; Hprt-R: 5’-ATCATCTCCACCAATAACTTTTATGT-3’. qRT-PCR was carried out in triplicated 20 µL reactions using SYBR Green PCR Master Mix (Applied Biosystems, UK), 1 µL cDNA, and primers at 600 mM using the ABI PRISM 7900HT instrument. PCR cycle was initial denaturation (95°C for 10 min), amplification and quantification repeated for 40 cycles (95°C for 45 s, 60°C for 30 s, and 72°C for 60 s with a single continuous fluorescence measurement), followed by a melting curve program (60–95°C, with a heating rate of 1°C per 30 s and a continuous fluorescence measurement). Mouse E15 placental cDNA was used to produce the standard curve. PCR products were identified by generating a melt curve, and results were expressed as mean Psg22 expression relative to mean Hprt expression.
Placental weights were measured after fixation in 4% paraformaldehyde (in phosphate buffered saline at pH 7.4). Samples were then dehydrated and embedded in paraffin wax. 5 mm thick sections were cut on a Leica RM2125RT microtome and stained with hematoxylin/eosin and viewed using an adapted Zeiss Jena Microfilm Reader fitted with a 17.5x projector lens. Simple point counting methods were used to estimate the volume fraction of spongiotrophoblast and the labyrinthine trophoblast compartments .
Statistical analyses were implemented in Microsoft Excel.