2.3. Lentiviral mediated reprogramming and induced pluripotent stem cell (iPSC) generation and characterization

Reprogramming was performed on DKO and control MEFs using a mouse STEMCCA lentiviral vector expressing Oct4, Sox2, Klf4, and cMyc. In brief, MEFs were grown in MEF media as previously described. Then 200 uL of STEMCCA vector and 1 uL of polybrene (Santa Cruz Biotechnology) were added to each well. After 24 hours, the cells were washed with DPBS and the media changed to ESC media DMEM supplemented with 15% FBS, 1 u/mL of leukemia inhibitory factor (LIF) (Millipore), 1% non-essential amino acids, and 1% penicillin/streptomycin. After 10–14 days, individual iPSC colonies were isolated and expanded on irradiated MEFs grown on gelatin-coated plates. We selected 4 individual iPSC clones from DKO MEFs and 4 individual iPSC clones from control MEFs.

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