Gene transfer in human monocyte-derived DCs with MPSV-NTP or pHR'NTP.

(A) Detection of vector sequence in DCs by Southern blot analysis. DCs were infected on day 4 at an MOI of 4 with the oncoretroviral vector MPSV-NTP (MPSV) or the lentiviral vector pHR'NTP (pHR'). Four days after infection genomic DNA was extracted and digested with eitherNheI (MPSV-NTP) or ScaI (pHR'NTP), which cut in the LTR. Southern blot analysis was performed using a radiolabeled NTP complementary DNA probe. The left 3 lanes represent copy controls obtained from MEL clones bearing 0, 1, or 2 copies of the MLV vector SFG-NTP digested with NheI. Position of the signals expected for the vector DNAs are indicated (copy control = 2.8 kb; MPSV = 1.8 kb; pHR' = 3.6 kb). EB indicates endogenous bands; −, untransduced; +, transduced. No band was observed in the MPSV⁺ lane, even after extended exposure (24 hours in Figure 3; 10 days for extended exposure). (B,C) Detection of integrated lentiviral vector DNA by Alu PCR. HeLa cells and DCs were either untreated (U) or infected with the HIV-based vector pHR'NTP (H) or the MLV-based vector MPSV-NTP (M). Proviral junctions were amplified using the primers 3′Alu and NTP3. PCR products were analyzed by gel electrophoresis (B) and Southern blot hybridization with an NTP-specific probe (C).