Molecular analyses of p210-transduced CB cells.

(A) Southern analysis of genomic DNA isolated from p210-tranduced CB cells 14 days after transduction after digestion with XbaI and hybridization with a GFP probe showing the expected full-length (13.6-kb) integrated BCR-ABL. (B) RT-PCR analysis of RNA isolated from MIG or p210-transduced CB cells 7 days after transduction showing evidence of BCR-ABL transcripts (upper gel) in the p210-transduced cells only and actin transcripts in all samples. Lane 1 contained no RT (negative control for RT-PCR). Lane 2 contained water (negative control for PCR). Lanes 3 to 5 contained amplified products from MIG-transduced CB cells (3 different experiments). Lanes 6 to 10 contained amplified products from p210-transduced CB cells (5 different experiments). Lane 11 contained amplified products from K562 cells (positive control). The expected sizes, in base pairs, are indicated for both PCR products. (C) Increased expression and phosphorylation of p210^BCR-ABL in p210-transduced CB cells as compared to K562 cells. A Western blot analysis of total cell lysates probed with an antiphosphotyrosine antibody is shown in the lower panel. The membrane was then stripped and reprobed with an anti-ABL antibody (upper panel). The positions of p210^BCR-ABL and the endogenous p145^c-ABL(endogenous loading control) are indicated. Total cell lysates from MIG-transduced CB cells and from the K562 cell line were used as negative and positive controls, respectively.