Quantitative analysis of vector DNA nuclear import in human CD34⁺ cells.

(A) Strategy for the determination of vector DNA status in human CD34⁺ cells. DNA from human CD34⁺-transduced cells was extracted 24 hours after the end of transduction and digested with EcoNI and AvaII. DNA was further digested with XhoI to minimize transfer bias due to the large size of circular DNA fragments. Southern blot was then performed using a PCR-generated DNA fragment exactly overlapping the EcoNI site as a probe. The internal 0.77-kb EcoNI-AvaII DNA fragment, common to all vector DNA species irrespective of their integrated or unintegrated state, indicates the total amount of reverse-transcribed vector DNA in transduced cells. The 1.16-kb and 1.4-kb signals correspond to unintegrated linear DNA and one LTR circular DNA, respectively. Due to the diversity of vector DNA integration sites, the sizes of digested genomic fragments containing the 5′-part of the vector DNA were very heterogeneous, and thus, the integrated vector DNA could not be revealed on the blot. Because the PCR-generated probe exactly overlaps the EcoNI site, the intensity of each signal is directly proportional to the amount of the corresponding vector DNA species. Thus, the amount of integrated proviral DNA can be calculated by subtracting the signals of unintegrated linear and circular vector DNA from the total amount of vector DNA. (B) Southern blot analysis of the vector DNA forms in CD34⁺-transduced cells. (C) Phosphorimage quantitation of intracellular vector DNA profiles in CD34⁺ cells transduced by TRIP or HR vectors. The results are expressed as percentages of total vector DNA. The data shown in panels B and C are the results of 1 representative experiment out of 3 independent experiments.