Retroviral Vector Transduction and Selection

Packaging of the pQCXIH and pQCXIH-ISceI retroviral vectors and infection of cell cultures was performed as previously described (40 (link)). The selection for cells infected with pQCXIH-ISceI was achieved by growth in medium containing 50 μg/ml hygromycin (Sigma) for 14 or 15 days, with medium changes every 2 days to allow for expression of I-SceI endonuclease and the generation of DSBs. After 13 days, the cells were trypsinized and replated. After an additional 1 or 2 days, the cells were trypsinized again, pooled, and either analyzed for the frequency of GFP-positive and DsRed-positive cells, or used for isolation of genomic DNA for analysis of small deletions.