DNA transfection using the Nucleofector
96-well Shuttle system (Lonza, Basel, Switzerland) was performed according to the manufacturer\\\\\\\\\\\\\\\'s instructions. Briefly, 6.25×105 cells were suspended with 20 µl of either of the supplied reagents (SE, SF
or SG), and transfected with 0.4 µg of linearized targeting vector, cultured for 20–24 h, and replated at a density of ~106 per 90-mm dish into agarose medium containing 0.4 mg/ml hygromycin B or
0.5 µg/ml puromycin. After a 2–3-week incubation, drug-resistant colonies were isolated and then subjected to further analysis