Figure 3. Effects of internal polyadenylation (poly-A) sites and transcriptional activity of ECOO on vector titers.

(A) Depiction of lentiviral vectors carrying poly-A signal sequences and internal promoters in different orientations. Arrows indicate the direction of transcription. BGH: bovine growth hormone poly-A signal; SV40: Simian virus 40 poly-A signal; pbi: tetracycline regulated promoter. (B) Bar graphs describing titers (TU/ml) and (C) physical particle concentration of the lentiviral vectors: pTK1588, pTK1595, pTK1848, pTK1850. Vectors were produced by transient transfection in 3 different 293T cell lines: naïve cells (black bars), β-globin knocked-down cells (white bars) and PKR knocked-down cells (grey bars). Titers of the conventional lentiviral vector pTK945 served as controls. Vectors were generated in 3 independent experiments (n=3), and represented as average ± SD. Titers (TU/ml) were determined by scoring GFP positive cells following serial dilutions on 293T cells. Concentrations of physical vector particles were determined by p24^gag ELISA. (D-E) Bar-graphs describing vector titers and concentration of physical vector particles of lentiviral vectors carrying a tetracycline inducible promoter and either SV40 or BGH poly-A signals, including pTK1885, pTK1927, pTK1936, pTK1937 The vectors were generated in naïve (black and diagonal black lines bars) and PKR knockdown (grey and diagonal grey line bars) 293T cells, either in the presence (diagonal lines containing bars) or absence of the tetracycline-regulated trans-activator, tTA (black and grey bars). The conventional pTK945 vector served as control. Vectors were generated in 3 independent experiments (n=3), and titer was represented as average ± SD. (D) Vector titers were determined by scoring GFP positive cells following serial dilutions on SODK0 cells, which constitutively express tTA. (E) Concentrations of physical vector particles were determined by p24^gag ELISA.