Template Switching by RNA Polymerase II In Vivo Eugene S Kandel, Evgeny Nudler  Molecular Cell  Volume 10, Issue 6, Pages 1495-1502 (December 2002) DOI: 10.1016/S1097-2765(02)00777-3

Figure 1 Experimental Scheme for Detecting the Hybrid Transcripts In Vivo The Moloney murine leukemia retroviral vector, pLNCX (Miller and Rosman, 1989), containing long terminal repeats (LTR), packaging signal (Ψ), and neomycin resistance marker (neo) serves as a template for PCR amplification with F1, R1 or F2, R2 pairs of primers. The resulting PCR fragments, A1 or A2, respectively, contain both provirus (pink) and plasmid (blue) derived sequences. RNAP II initiating transcription within the 5′ LTR (+1 stands for the transcription start) of A1 or A2 incorporates the sequence of the second LTR into the transcript by switching over to the 5′-end of the same (right) or identical (left) template molecule. In either case, reverse transcription results in a provirus that embodies sequences of the plasmid into its genome (bottom). Molecular Cell 2002 10, 1495-1502DOI: (10.1016/S1097-2765(02)00777-3)

Figure 2 Analysis of Recombinant Proviruses (A) PCR amplification with primers L and R of A1 or A2 proviruses of Figure 1 is expected to yield 255 bp A1P or 256 bp A2P products, respectively, with characteristic sequences around the junction points. (B) Confirmation of the predicted structure of A2P by restriction enzyme digestion and fluorescent sequencing . The autoradiogram of 6% PAGE urea gel shows end-labeled A2P and A1P products before (lanes 2 and 4) and after (lanes 3 and 5) treatment with 5 units of Drd I for 10 min at 37°C. 100 bp DNA ladder was end-labeled and used as a size marker (lane 1). The sequencing of both strands of A2P was performed on ABI PRISM Model 377 using L and R oligonucleotides as primers. Molecular Cell 2002 10, 1495-1502DOI: (10.1016/S1097-2765(02)00777-3)

Figure 3 Control for the A2 Template Ligation in the Packaging Cells (A) Experimental protocol. pLNCX2R was constructed by substituting the original BamHI-NruI fragment for the double-stranded oligonucleotide (insert). This creates a new binding site for the R primer. Both Template A2 and pLNCX2R (the latter was diluted 103-fold with salmon sperm DNA) were transfected into PhoenixEco packaging cells using the Lipofectamine Plus reagent (Invitrogen) followed by DNA recovery and PCR analysis. (B) PCR analysis of DNA from packaging cells with the R and L primers. 2% agarose gel (left panel) shows a 214 bp product (lane 3) generated after 27 cycles (58°C annealing temperature) of PCR on DNA from pLNCX2R transfected cells. Under the same conditions no PCR product could be detected from DNA of untransfected PhoenixEco cells (lane 1) or cells transfected with A2 (lane 2). M, marker. (C) Drd I restriction analysis. Ethidium bromide stained 10% PAGE shows DNA products generated after 36 cycles of PCR with primers L and R on DNA from PhoenixEco cells transfected with A2 (lanes 3 and 4), and also from HT1080 cells infected as in Figure 2 (lane 2). PCR products (0.25 μg) in lanes 2 and 3 were digested with DrdI. Only the PCR product from infected HT1080 cells was sensitive to DrdI (lane 2). M, marker. Molecular Cell 2002 10, 1495-1502DOI: (10.1016/S1097-2765(02)00777-3)

Figure 4 Testing the Role of the LTR Promoter in the Switch-Over Product Formation Templates “LX” and “ΔLX” were prepared by PCR from pLXSN and pΔLXSN retroviral vectors, respectively, that contained an internal SV40-derived promoter (SV40), neomycin resistance gene (neo), and packaging signal (Ψ). ΔLX template carries a deletion in the LTR promoter/enhancer region. Upon transfection into the packaging cells, the LTR of the ΔLX template remains nonfunctional as a RNAP II promoter. Transduction of neo gene to the target HT1080 cells may be monitored via G418 selection and PCR with NEOS and NEOAS primers, while the predicted switch-over product should contain the annealing sites for primers DRDS and DRDAS. HT1080 cells were infected with viral supernatants generated upon transfection of PhoenixEco cells with either LX or ΔLX templates. Infected cells were G418-selected and their DNA was extracted to obtain LX and ΔLX samples. Both samples are positive for the neomycin resistance gene (top and bottom panels, lane 2). However, the switch-over product is found only in the LX sample (top panel, lane 3) but not ΔLX (bottom panel, lane 3). Lane 1 is the water control. Molecular Cell 2002 10, 1495-1502DOI: (10.1016/S1097-2765(02)00777-3)