Figure 2

Design of the SINIL-2pr retrovector. A. pGFP/TKfus plasmid bears a full-length 3'LTR that incorporates all the promoter and enhancer machinery intrinsic to the wild-type MoMLV retrovirus. The CMV promoter element which substitutes the U3 region in the 5' LTR drives the expression of the retroviral genome in transfected packaging cells for the production of replication-defective retrovirus. B. SINIL-2 pr retrovector is designed by creating an NheI-AscI deletion to the 3'LTR of pGFP/TKfus and by replacing the U3 with the full-length human IL-2 promoter which results in a self-inactivating retrovector whereby expression of the EGFP and HSVTK fusion protein is dependent on the IL-2 promoter in cells transduced with SINIL-2pr retroparticles. C. Southern Blot analysis of the integrated proviral DNA in Jurkat and p116 cells transduced with SINIL-2pr retrovector at similar MOI. Genomic DNA was extracted from transduced and control null cells, digested with KpnI and probed with [P³²]-labeled probe complementary to the GFP reporter cDNA. The detected DNA bands are consistent with the predicted sized fragment indicating no rearrangement in the integrated proviral DNA.