Generating Mouse Embryonic Stem Cells

Puromycin-resistant MEF feeder cells (Cell Biolabs, CBA-312) and neomycin-resistant MEF feeder cells (Cell Biolabs, CBA-311) were cultured in DMEM high glucose supplemented with 10% fetal bovine serum and 1% PenStrep at 37°C, then inactivated with 10 µg/ml mitomycin C (Sigma, M4287) for 2 hr for mouse embryonic stem cell culture, as previously described (Hai et al., 2018 (link)). DBA/2J mouse ES cell line AC173/GrsrJ (JAX, 000671C02), was cultured on 0.1% gelatin-coated plates with mitomycin C inactivated MEFs, in ES culture medium (DMEM high glucose with 15% fetal bovine serum, 1% MEM Non-Essential Amino Acids, 1% PenStrep, 0.1 mM 2-mercaptoethanol, 10³ unit/ml leukemia inhibitory factor (Millipore Sigma, ESG1107), 1 μM PD0325901 (Sigma, PZ0162) and 3 μM CHIR99021 (Sigma, 361571)), at 37°C. All cell lines were detached with trypsin and frozen with 80% ES culture medium supplemented with 10% DMSO and an additional 10% FBS. Only mouse cell lines used. No STR profiling methods are available to authenticate mouse cell lines. DNA sequencing was used to confirm editing of the DBA/2 allele to the AKR allele of the mouse ES cell line used. Mycoplasm testing was negative.