Figure 1. Drawings of the self-inactivating, insulated pFIN lentiviral vector and the dual promoter proviral DNA and its associated mRNA transcripts

A: The pFIN lentiviral vector (8101 bp) contains viral sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes necessary for self-replication. Modifications made to the 5' and 3' LTRs that insure that the virus is unable to replicate [32], and the addition of the cPPT-FLAP sequence to the vector that improves viral transduction (10) have been previously described. In this study, we inserted the 2x250 chicken β-globin HS4 insulator sequence into the deleted U3 (dl.U3) region of the 3'LTR of the vector to improve expression of the integrated transgene. Transgenes were cloned into the multiple cloning site located between the cPPT-FLAP and HS4 insulator sequences. The pFIN vector carries the β-lactamase gene that confers ampicillin resistance (Amp^r). B: Reverse transcription of the RNA genome of the virus following infection of the cell into double-stranded DNA generates proviral DNA in which the transgene(s) is flanked by the HS4 insulator sequences. In our dual promoter vectors, the two transgenes that we insert into the multiple cloning site share the same polyadenylation signal that is located in the deleted 3'-LTR region. Because the two transgenes share the same polyadenylation signal, the transcript encoding gene 1 is longer and includes the promoter and coding regions of gene 2 in the 3' untranslated region of the transcript. Vector elements: CMV-TATA-TAR-recombinant cytomegolovirus (CMV) immediate early enhancer/TATA promoter that replaced the 5' U3 of the long terminal repeat (LTR); dl.R, U5 and U3-deleted LTR sequences; partial gag-partial sequence of the viral gene encoding the matrix and core group antigens; RRE-Rev responsive element; cPPT-FLAP-central polypurine tract/DNA FLAP sequence; MCS-multiple cloning site; HS4-chicken β-globin insulator; bGHpA is the bovine growth hormone polyadenylation sequence that replaced the 3' U5; Prom 1 and 2-transgene promoters.