Cortical astrocytes were purified from neonatal H-2KbtsA58 transgenic mice as described above, except that the cultures were prepared at 33°C in the presence of 25 U/mL of IFN-y. The cultures were passaged at a ratio of 1:4 and refed. The following day, cultures were incubated with 1 mL of BAG retroviral supernatant (Price et al., 1989), 1 mL of DMEM-FCS and 2 mL of Polybrene (10 mg/mL; Sigma) for 2 h at 37°C. The cultures were then washed with DMEM-FCS, fed with DMEM-FCS containing 25 U/mL of IFN-y, and shifted back down to 33°C. The cells were passaged at a ratio of 1:4 once more the following day and fed with the same medium. After 4 d, the culture medium was supplemented with 250 mg/mL G418 (Geneticin; Gibco-BRL) and the cultures fed every 3 d for 2 wk, after which time drug-resistant colonies were clearly visible. Fifty colonies were passaged using autoclaved cloning rings and cultured in 96-well trays. The cell lines were expanded progressively to 24-and 6-well trays, and then to T25 and T75 tissue-culture flasks. The passage at which the lines were plated into a T25 flask was designated as Passage 1. Ten lines were chosen for Southern blot analysis using a 1.1-kb probe for the neo gene to determine whether each line contained a single retroviral integration site.
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