Curred in the processing of the Gag precursor in the presence
Curred in the processing of the Gag precursor in the presence of Vif, although we detected an improvement in the processing of Vpr-RT/IN with the 6 plasmid system, as can be seen in Figure 4 by the concurrent reduction in Vpr-RT/IN and increase in RT (lanes 3 and 4).Self-inactivating (SIN) get 11-Deoxojervine vector improves viral titers In addition to modifying the packaging system, we also constructed a SIN lentiviral vector to improve further the safety of the system by decreasing the risk of provirus mobilization and RCL formation. This SIN vector was constructed by modifying the U3 and U5 regions of the 3′ LTR, as follows: a 400 bp deletion was created in the U3 region between the EcoRV to the Pvu II restriction sites to remove viral enhancer and promoter, and the U5 region was entirely eliminated and replaced by an “ideal” termination/polyadenylation sequence (ATG TGT GTG TTGFigure 5 wt versus T26S mutant Comparison of titers produced by PR expression plasmids: Comparison of titers produced by PR expression plasmids: wt versus T26S mutant. NIH 3T3 cells were infected with serial dilutions of viral supernatants produced by the 7 plasmid system with either the wt (blue) or T26S mutant (red) PR. Titers were determined by monitoring transduced NIH 3T3 cells for the production of GFP by FACS. In this study, the lentiviral vector (wt-LTR expressing GFP), Rev, Tat, VSV-G, Gag (non-optimized), and Vpr-RT/IN DNA amounts remained constant, while the DNA amounts of Vpr-wt PR (wt protease, shown in blue) and Vpr-T26S PR (mutant protease, shown in red) varied. Experiments were performed using two concentrations for Gag and Vpr-RT/IN: (1? using 1.3 g Gag and 2.3 g Vpr-RT/IN DNA with varying amounts of PR DNA (0.7 g, 1.0 g, 1.3 g, and 1.6 g), indicated on the graph by circles (l), and (2? using 2.6 g Gag and 4.5 g Vpr-RT/IN DNA along with varying amounts of PR DNA (0.8 g, 1.4 g, 2.0 g 2.6 g, 3.2 g), indicated on the graph by triangles (s). In these initial studies the VprRT/IN plasmid did not contain Vif, the functional titers ranged from 0.4 ?105 TU/ml to 3.0 ?105 TU/ml. N = 1.GTT TTT TGT GT). In addition, two stop codons were also introduced within the region where the packaging signal and Gag overlap, so that Gag could not be reconstituted if a recombination occurred and to prevent the translation of a residual Gag peptide. The remaining portions of this vector are identical to those of the wt-LTR lentiviral vector, that is, they both contain an unmodified 5′ LTR (so that the lentiviral vector remains Tat dependent), the central polypurine tract, RRE, and an Ef1 promoter driving GFP expression (Fig. 6A). In conjunction with the SIN vector we chose to continue to supply Tat in trans due to safety concerns, that is to say, since the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 5′ LTR in our vectors do not contain a strong promoter (such as CMV or RSV) and still require Tat to properly activate their HIV-1 promoter,Page 8 of(page number not for citation purposes)Retrovirology 2007, 4:http://www.retrovirology.com/content/4/1/than the Tat transactivation of the promoter acts as a safeguard preventing the production of full length packagable transcripts by the integrated vector. To determine if this SIN vector would significantly affect titers, 293T cells were transfected with plasmids composing each of the three packaging PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28212752 systems in conjunction with the SIN vector (Fig. 6B), the resulting supernatants were then used to transduce NIH 3T3 cells. Titers were determined by FACS analysis for the expression of GFP. F.
