mtna2014 Sup info (original) (raw)

Legend to supplementary figures. Supplementary figure S1 Titers of pTrip PGK-Pac/CMV-Gfp vector stocks with different IN, assessed as measurements of p24/ul, vRNAc and TU/ul. Thirty stocks of vectors were analyzed to determine their content in viral capsid protein p24, in viral RNA copies (vRNAc) and transducing units (TU) per volume of supernatant. (a) The content of p24/ul of supernatant does not vary between stocks of vectors carrying different IN. (b) Although fluctuating, the content of vRNAc/ul was not statistically different in the group of vectors analyzed. (c) No significant differences in TU/ul are found between the different stocks of vectors. (d) No statistical differences are found between groups of vectors when comparing values of the ratio TU ml-1 /p24 ml-1. (e) Significant differences are measured when comparing the ratio TU/vRNAc, with higher values for D167H vectors as compared to any of all other vector types. Error bars represent mean+SEM. Statistics, One way ANOVA and Tuckey pot hoc test; (NS) p > 0.05; (*) p< 0.05; (**) p< 0.01; (***) p < 0.001. Supplementary figure S2 Analysis of pTrip PGK-Pac/CMV-Gfp transduction efficiencies in HEK-293T cells with vectors bearing different integrases with respect to time and vector input. (a) Percentage of GFP+ cells transduced with 10 vRNAc of vectors Q168A, D64V, N and LQ at different times after transduction as assessed with FACS. (b) Comparison of GFP MFI, at day 21 after transduction, in HEK-293T cells incubated with increasing M.O.I. of pTrip PGK-Pac/CMV-Gfp vectors carrying different IN mutations. At M.O.I. 300 and 900, MFI is higher in cells transduced with D167H as compared to that transduced with WT while MFI remains steady with vectors Q168A, D64V, N and LQ. At M.O.I. of 2700 vRNAc, MFI of vectors WT and D167H are equivalent sugesting toxicity associated to overtransduction with vector D167H. (c) GFP expression in HEK 293T cells transduced M.O.I. of 300 vRNAc with vectors Q168A, D64V, N and LQ at different time points after transduction; note that at day 7 after transduction all IDLV vectors allowed transducing nearly 100 % of the cells. Experiments (a) were done 3 times in duplicate with different stocks of vectors. Experiments (b) and (c) were done twice in duplicate. Error bars represent mean+SEM. Statistics: in all experiments two way ANOVA and Bonferroni's post-hoc test; (NS) p > 0.05; (*) p< 0.05; (**) p< 0.01; (***)p < 0.001. Supplementary figure S3 Comparison of transduction efficiency and integration rate of different IDLV in HEK-293T cells after transduction with increasing input of vector pTrip PGK-Pac/CMV-Gfp. (a) Percentage of GFP+ cells 4 days after transduction with 100, 300, 900 or 2700 vRNAc/cell of vectors carrying IN Q168A, D64V, N or LQ. (b) Number of puromycin resistant colonies counted 2 weeks after transduction with 100, 300, 900 or 2700 vRNAC/cell of vectors carrying IN Q168A, D64V, N or LQ. Experiments were done twice in duplicate. Non-transduced cells all died in puromycin medium. Error bars represent mean+SEM. Statistics: two way ANOVA and Bonferroni posttest, (NS) p > 0.05; (*) p< 0.005; (**) p< 0.01; (***) p < 0.001.