Ppackh1

NZB morula-blastocyst stage embryos were cultured 24 hours in KSOM medium. The zona pellucida was removed with acidic Tyrode’s solution (Sigma-Aldrich) and blastocysts were individually plated onto mitomycin C (Sigma-Aldrich) treated mouse embryonic fibroblast (mEF) feeder layers in ES derivation medium: KODMEM (Invitrogen) containing 1mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 100 μM β-mercaptoethanol (Sigma), 100 μM nonessential amino acids (Invitrogen), 1,000 units/mL LIF (Millipore), 10% FBS and 10% KSR (Invitrogen). ICM outgrowth colony was collected and dispersed into single cells using 0.15% trypsin solution (Sigma-Aldrich) before replating onto fresh mEF in ES medium containing 1 μM PD0325901 (Axon) and 3 μM CHIR99021 (Axon).
To induce fluorescent marker uniformly expressed under the control of the Ubiquitin-C promoter in NZB ES cell lines, Ubc-Td-tomato coding sequence was cloned into lentiviral vector with pPACKH1 (SBI) and Purfection (SBI) (Shibuya et al., 2003 (link); Yamaguchi et al., 2012 (link)). The presence of Ubc-Td-tomato expression was examined by fluorescence under 568 nm light and confirmed by FACS sorting purification (BD AriaII). GFP labeled B6 ESC line carrying mtDNA mutations, were generated using PolG mutator mice and B6 Ubc-GFP mice (Jackson laboratory) as recently described (Ma et al., 2020 (link)).