Hypoxanthine aminopterin thymidine supplement

All experiments used the E14Tg2a.4 ^{40 (link)} male mouse ES cell line, which has a 36 kb X chromosome deletion that removes the first two exons of the Hprt gene. Cells were grown under feeder free conditions on gelatin coated plates and fed standard ES cell medium: Knockout DMEM (Life Technologies) containing 15% fetal bovine serum (HyClone), 2 mM L-glutamine (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), 0.05 mM 2-mercaptoethanol (Sigma), 1,000 U/mL ESGRO® LIF (Millipore), and penicillin-streptomycin. Cells were fed daily. Approximately 20 ug of linearized plasmid DNA was transfected into 1–1.5×10⁷ ES cells in 0.8 mL HEPES buffered saline (Sigma-Aldrich) using a Gene Pulser Xcell™ (Bio-Rad) set to 250 V and 500 μF. Ten such transfections were performed for each library. Correctly targeted cells were selected by the addition of Hypoxanthine-aminopterin-thymidine (HAT) Supplement (Life Technologies) to the ES cell medium for 3–10 days, beginning 24 hours after transfection. Following HAT selection, cells were fed ES cell medium containing 1× HT supplement (Life Technologies) for two days. Cells from the same library were pooled together and expanded on fresh plates prior to sorting.