Generating Inducible Cre Expressing mES Cells

Mouse embryonic stem (ES) cells were cultured on irradiated MEFs in DMEM / 15% FBS, penicillin/streptomycin (P/S, Gibco), 2 mM glutamax (Invitrogen), nonessential amino acids, 0.1 mM β-mercaptoethanol, and 100 U/mL LIF (Peprotech). For EB differentiation, ES cells were trypsinized, and re-plated in differentiation medium (IMDM/15% FBS, 200 μg/mL transferrin (Sigma), 4.5 mM monothiolglycerol (MTG, Sigma), 50 μg/mL ascorbic acid (Sigma), and 2 mM glutamax) for 30 min to allow MEFs to adhere. Nonadherent cells (10⁵) were plated as a cell suspension in low adherence dishes on a slowly rotating shaker. To generate A2Lox.cre ES cells, the HPRT 5′ repair/targeting plasmid [9 (link)] carrying the cassette exchange TRE-2loxP-Δneo inducible target locus [10 ] was digested with XhoI and ligated to an XhoI-SalI fragment bearing the cre transgene from pSalk-cre [11 (link)]. 20 μg of SalI-linearized DNA was electroporated into 6×10⁶ A17 mES cells, and selection in ES medium with HAT supplement (Invitrogen) was initiated 24 hours later.