CHO DG44 Cell Culture and Transfection

CHO DG44 cells, obtained from Dr. Lawrence Chasin at Columbia University, were maintained in α-MEM medium with nucleotide (Sigma, M8042) supplemented with 10% fetal bovine serum (FBS), as previously described [8] (link), [14] (link). Plasmid DNA was transfected into cells using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM (Gibco). One day after transfection, 5 µg/ml of blasticidine was added to select for transformants, and the concentration was increased to 10 µg/ml on day 10. Usually, on day 23, the medium was replaced with α-MEM without nucleotide (Sigma, M4526) supplemented with 10% dialyzed FBS (Thermo Scientific). Methotrexate (Sigma) was dissolved in water and added to the culture at 5 or 50 nM. Cloning of cells was performed by limiting dilution.