Male dba 2 mice

Female CBA/J mice (RRID : IMSR_JAX:000656), male DBA/2 mice (RRID : IMSR_JAX:000671), male BALB/cJ mice (RRID : IMSR_JAX:000651) and wildtype C57BL/6NJ mice (RRID : IMSR_JAX:005304) were purchased from Jackson Laboratory. After adaptive feeding, Female CBA/J mice were mated to male BALB/c or DBA/2 mice to establish the models of normal pregnancy (CBA/J × BALB/c, n=5) or spontaneous abortion (CBA/J × DBA/2, n=6). The fertilized eggs of C57BL/6 mice were microinjected with Cas 9/guide RNA complexes targeting at exons 2, 3, and 4 of the murine Pgf, incubated in a 5% CO₂ incubator at 37°C until the two‐cell stage, and then transferred to foster mothers. Then the F1 heterozygotes were generated by the backcrossing of genome‐modified F0 generation mice with wild‐type mice, and confirmed by qRT-PCR. To avoid the confounding effect of embryonic genotype, the heterozygotes were mated with wild-type mice to generate pregnant mice in the experimental group (Pgf^-/+ females × wild-type males, n=24) and control group (wild-type females × Pgf^-/+ males, n=26).

Male DBA/2 mice were purchased from The Jackson Laboratory Japan Inc. (Yokohama, Japan). Mice were 6 weeks of age at the time of the experiment. All the procedures were approved by the Committee for Animal Experiments of Kobe University School of Medicine and performed under the Guidelines for Animal Experiments. KLN205, which was obtained from Riken Bioresource Center, is an established cell line of SCC generated from a DBA/2 mouse. KLN205 cells were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum, penicillin G potassium, and 0.1 mM non-essential amino acids. They were maintained at 37 °C in a 5% CO₂ humidified incubator. Cells were passaged two times a week into fresh growth medium.