*4.2. Animals and Sampling*

Stab1/2 dKO mice were generated by crossing Stab1 KO mice with Stab2 KO mice [8,21]. Male and female Stab1/2 dKO mice were intercrossed to generate Stab1/2 dKO embryos and placentas (Figure 2). The mating of males and females for pregnancy was performed at the ratio of 1:1 in a single cage; and 2 weeks after mating, the females were separated into different cages and followed until birth or pregnancy loss (observation for at least 20 days after separation from the males). The date of pregnancy of the pregnant females was determined by the normal gross fetal anatomy of embryos obtained in their uteri and the comparison based on the text "The atlas of mouse development (edited by M. H. Kaufman, Elsevier academic press)". The genotypes of mice and/or embryos were analyzed using PCR and genomic DNA isolated from tails or yolk sacs. Primer sets and PCR amplification conditions for Stab1 and Stab2 were previously described [8,21]. WT females or embryos were used as controls. The mice were bred and maintained in climate-controlled (20–25 ◦C), specific pathogen-free conditions with a 12 h/12 h light/dark cycle and were allowed free access to standard mouse diet and water. All procedures concerning animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Kyungpook National University (Approval number KNU-2016-0011, Daegu, Korea).
