Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes-0

in pCDNA3. The RRE from HIV-1 (350 nt) or from SIVmac239 (272 or 1045 nt) was positioned downstream of the Gag/Pol coding sequence. Polyadenylyation sequence in pCDNA3 is derived from bovine growth hormone gene (BGHpA). B) The gene transfer vectors were derived from pNL4-3 and contain a transgene expression cassette consisting of Elongation factor 1 alpha promoter/enhancer elements (EF1α) driving the enhanced green fluorescent protein (EGFP) or a fusion protein consisting of EGFP-2A-Rev M10. Woodchuck post-transcriptional regulatory element (WPRE) was positioned downstream of the transgene. HIV-1 or SIV RRE was present upstream of the transgene expression cassette. Δψ: Deletion in the HIV-1 encapsidation signal between nt 751 and nt 779 of pNL4-3; LTR: HIV-1 long terminal repeat; FS: Frame-shift mutation in ; CPPT/CTS: Central polypurine tract/central termination sequence; 2A: Foot and mouth disease virus 2A cleavage factor; M10: Rev M10; 5'ss: 5' splice site; 3'ss: 3' splice site.<p><b>Copyright information:</b></p><p>Taken from "Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes"</p><p>http://www.aidsrestherapy.com/content/5/1/11</p><p>AIDS Research and Therapy 2008;5():11-11.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2438438.</p><p></p

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes-3

RE. The SIV RRE-based packaging system (SIV RRE system) consisted of the packaging plasmid pGP/SIV 1045 RRE and the gene transfer vector pN-EF1α-EGFP/SIV RRE. For production of virus stocks with the SIV RRE-based packaging system either HIV-1 Rev (pCI-HIV Rev) or SIV Rev (pCI-SIV Rev) expression construct was used, as indicated. All transfections also received a VSV-G envelope expression construct (pMD.G) and a HIV-1 Tat (pCMVtat) expression construct. The titers of the vector stocks were determined as described in Materials and Methods. The % of GFP + cells in the absence of pCI-Rev M10 (0 μg) was considered as 100% (Y-axis) for a given packaging system to which other titers obtained at each amount of pCI-Rev M10 were normalized. Increasing amounts of pCI-Rev M10 are depicted on the X-axis. For each transfection, the indicated amount of pCI-Neo was used as a 'filler', to keep the total amount of DNA added at 1.0 μg. The results shown are representative of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes"</p><p>http://www.aidsrestherapy.com/content/5/1/11</p><p>AIDS Research and Therapy 2008;5():11-11.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2438438.</p><p></p

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes-6

refers to the length of SIV RRE present in the packaging construct or gene transfer vector. The effect of expression of Rev-like proteins from HIV-1 (pCI-HIV-Rev), SIV (pCI-SIV-Rev) and HTLV-1 Rex (pBCRex-1) on vector stock production was tested with each of the combinations. A control vector, pCI-Neo, was used in parallel. The mean vector titers, shown in the top panel, were calculated from % GFP positivity determined by flow cytometry. The bottom panel depicts mean p24 levels in the vector containing supernatants. The titers and p24 levels were normalized to SEAP activity present in the vector stock. Error bar = 1 SD. IU: infectious units. '*' denotes relatively high p24 levels with respect to titer (described in greater detail in the text).<p><b>Copyright information:</b></p><p>Taken from "Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes"</p><p>http://www.aidsrestherapy.com/content/5/1/11</p><p>AIDS Research and Therapy 2008;5():11-11.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2438438.</p><p></p

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes-5

in pCDNA3. The RRE from HIV-1 (350 nt) or from SIVmac239 (272 or 1045 nt) was positioned downstream of the Gag/Pol coding sequence. Polyadenylyation sequence in pCDNA3 is derived from bovine growth hormone gene (BGHpA). B) The gene transfer vectors were derived from pNL4-3 and contain a transgene expression cassette consisting of Elongation factor 1 alpha promoter/enhancer elements (EF1α) driving the enhanced green fluorescent protein (EGFP) or a fusion protein consisting of EGFP-2A-Rev M10. Woodchuck post-transcriptional regulatory element (WPRE) was positioned downstream of the transgene. HIV-1 or SIV RRE was present upstream of the transgene expression cassette. Δψ: Deletion in the HIV-1 encapsidation signal between nt 751 and nt 779 of pNL4-3; LTR: HIV-1 long terminal repeat; FS: Frame-shift mutation in ; CPPT/CTS: Central polypurine tract/central termination sequence; 2A: Foot and mouth disease virus 2A cleavage factor; M10: Rev M10; 5'ss: 5' splice site; 3'ss: 3' splice site.<p><b>Copyright information:</b></p><p>Taken from "Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes"</p><p>http://www.aidsrestherapy.com/content/5/1/11</p><p>AIDS Research and Therapy 2008;5():11-11.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2438438.</p><p></p

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes-4

Fected or infected with VSV-G pseudotyped pNL4-3.HSA. RE. The supernatants from mock or virus-infected cells were obtained on days 1, 4 and 7 and assayed for HIV-1 p24 capsid protein using a commercial ELISA kit. The Y-axis shows mean p24 levels produced by each of the different cell populations on days 4 and 7 normalized to the p24 produced by infected but untransduced Jurkat cells (which was set at 100%). The results shown are from three independent experiments. Error bar = 1 SD.<p><b>Copyright information:</b></p><p>Taken from "Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes"</p><p>http://www.aidsrestherapy.com/content/5/1/11</p><p>AIDS Research and Therapy 2008;5():11-11.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2438438.</p><p></p

Validation of GD method using predefined amplicon mixes.

<p>High-resolution melt curves of samples containing different proportions of F8-S3 amplicon (S3Wt) or F8-S2 mutant (S2Mt) in F8-S2 amplicon (S2Wt) were analyzed by GD as detailed in Materials and Methods. (A) Derivative melt curve data (blue dots) of indicated S3Wt-S2Wt mixes were fitted using 3-GD (red traces). The nominal percentage of S3Wt in the mix is shown below (indicated by S3Wt%) and the GD-estimated amount in the top left corner of each plot. (B) Derivative melt curve data (blue dots) of indicated S2Mt-S2Wt mixes were fitted using 3-GD (red traces). The nominal percentage of S2Mt in the mix is shown below (indicated by S2Mt%) and the GD-estimated amount in the top left corner of each plot. (C) Scatter plot of nominal F8-S3Wt% in mix (X-axis) vs 3-GD estimated F8-S3Wt% (Y-axis). (D) Scatter plot of nominal F8-S2Mt% in mix (X-axis) vs 3-GD estimated F8-S2Mt% (Y-axis). The equations of linear regression analysis of both types of dose-response curves and the correlation coefficients (R²) are shown. The samples were tested in duplicate (replicates ‘a’ and ‘b’).</p

Size of PCR product does not affect determination of mutant percentage by GD.

<p>The CCR5 target site in gDNA of unmodified or genome-edited cells were amplified using two pairs of primers designed to produce two distinct sizes of product (107 bp and 140 bp, respectively). The amplicons were subjected to high-resolution melting and then processed to correct for temperature-dependent quenching of fluorescence of free and dsDNA-bound fluorophore. The resulting melt curves of genome-edited (for clone pair L1R1) and unmodified controls (Mock) are shown (A & C). Corresponding first-derivatives of processed melt curves are shown in B and D. Replicates G1 and G2, A1 and A2 refer to gDNA samples amplified using primers that produce 107 bp amplicon, whereas G5 and G6, and A5 and A6 refer to gDNA samples amplified using primers that produce 140 bp amplicon. The derivative melt curves were decomposed using the 3-GD model to estimate the mutant frequency. The estimated mutant frequencies for both sizes of amplicons are shown in (E). Error bar = 1 SD.</p

2-GD model shows better fit than 1-GD for derivative melt curve data of mocks.

<p>2-GD model shows better fit than 1-GD for derivative melt curve data of mocks.</p

Analysis of F8-S2 and CCR5 target sequence features and melting properties <i>in silico</i>.

<p>Sliding window analysis of percentage of AT (%AT) in F8-S2 (A) or CCR5 (B) sequences of target sites amplified by PCR. The percentage of As and Ts were determined in a sliding overlapping window of 10-mers. The shift was by 1 bp. These are shown as green dashes. The data was smoothed using running averages with a period of 5 (solid green line). The sum of free energies (∆Gs) in a sliding window of 10-mers and a shift of 1 bp is shown along the left y-axis in kJ/mol (blue dots). The running averages were calculated as for %AT traces and are shown as blue traces. Putative AT-rich domains are marked I-IV. (C- H) The F8-S2 and CCR5 target sequences were used as input in the UMelt web analysis tool (29). UMelt predicted derivative melt curve (C and D), "Dynamic Profile” of melting (E and F) using a sliding temperature control that was situated close to the predicted Tm for each sequence to identify portions of the target sequences (nucleotide position indicated on the x-axis) that may have melted earlier than the rest. The web tool also provided a "Melting Profile" analysis that shows potential regions that might show greater tendency to melt earlier (G and H).</p

Comparison of mutant percentage estimation by 2- and 3-GD.

<p>First derivatives of high-resolution melt curves from genome-edited samples were curve fitted using 2- or 3-GD models as described in Materials and Methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190192#pone.0190192.e012" target="_blank">Eq 11</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190192#pone.0190192.e013" target="_blank">Eq 12</a>, respectively). The mutant percentages estimated from curve fitting are shown along the y-axis for F8-S2 (A) and CCR5 (B). Two molecular clones (10 and 11) of dgRNAs targeting F8-S2 site and two pairs of TALENs (L1R1 and L2R2) targeting CCR5 site were tested. The mutant percentages were compared using Student’s t-test (two-tailed). The p-values of the pair-wise comparisons of 2-GD and 3-GD are shown above the bars.</p