Hylorondase

5IU of PMSG (Prospec Cat# HOR-272) and HCG (Sigma-Aldrich, Cat # CG5–1VL) were injected intraperitoneally into 4–5 week old, wild-type C57BL/6J female or or C57BL/6J C3H/HeJ F1 mice 46–48h apart. Immediately following HCG injection, females were paired with C57BL/6J stud males and pronuclear stage embryos together with cumulus cell clusters were harvested from plugged females 20h after the mating. Pronuclear stage embryos were dissociated from cumulus cells using Hylorondase (Fisher, Cat # MR-0510F). Embryos were then incubated at 37C with 5% CO2 while siRNAs were prepared for injection. Before injection, scrambled siRNA (Thermo Fisher Scientific, Cat# AM4611) and Klf5 siRNAs (Thermo Fisher Scientific, Cat# 160900) and Klf4 siRNAs (Thermo Fisher Scientific, Cat# 156021) were prepared at a working concentration of 100uM were spun at 10k rpm for 5 min to clear debris. In experiments when we double knocked down Klf4 and Klf5, we prepared siRNA mixtures containing 50 uM Klf4 siRNAs and 50 uM Klf5 siRNAs. After the microinjection of siRNAs, embryos were cultured in vitro to E4.0, and then subjected to IF analyses described above.

C57BL/6J or C57BL/6J C3H/HeJ F1 mice were crossed to C57BL/6J stud males and pronuclear stage embryos were dissociated from cumulus cells using Hylorondase (Fisher, Cat # MR-0510F). After the zona was weakened with acid Tyrode’s (Sigma Aldrich, Cat # T1788), the embryos were subsequently washed in M2 buffer. Cas9 RNP complexes were then assembled in vitro by combining 40uM of Cas9 protein with 2ug of sgRNAs targeting Klf5 (CCAGACCGUCCAUGCCCACG, AGCACCCGCGUGGGCAUGGA, GGUC AGCACCCGCGUGGGCA, Synthego). Assembled RNPs were then mixed with a cohort of 50–75 embryos, and electroporated with standard parameters (Modzelewski et al., 2018 (link)). Electroporated embryos were then cultured in KSOM until E3.5, at which point they were processed for IF analysis described above.