Genetic analyses of insertional mutants obtained after step 2. (A) Representative Southern blot analysis using 10 μg genomic DNA of expanded clones. Only selected clones of each experiment are shown displaying a different band pattern within each blot. The cDNA for eGFP or DsRed or a fragment spanning the wPRE were used for probing. Clone names are indicated above each lane. (B) Location of insertions into the Evi1 allele. The Mds1 locus lies further upstream in the same transcriptional orientation. LTR and SIN vectors recovered in our in vitro studies show the same pattern, consistent with our previous findings made with LTR vectors in dominant or leukemic clones in vivo.^7,8,12  (C) Quantitative real-time PCR was used to determine the transcript level of Evi1 or HoxA7. Bars represent the relative enhancement compared with expression levels in mock-transduced and expanded cells. Clone 6.4 contains a vector insertion upstream of HoxA7, whereas clone C4 does not. Each PCR was performed in triplicate, and bars represent the mean of 3 CT values. Values for clones 1.5 and a1 represent the average of 2 independent determinates performed in triplicate.