Activities of individually expressed shRNAs from the same vector. (a) 400 ng each of a series of 1, 2, 3 and 4 cassette (ca) pLenti6 (pL) based vectors with only a single hairpin per vector (surrounded by 'empty' expression cassettes) were assayed with 300 ng each of T.r (target-specific) and V.r-red (control) (n = 3). (b) 0 - 400 ng each of a series of 1×, 2×, 3× and 4× cassette pL vectors with all cassettes containing a T shRNA were assayed with 300 ng each of T.r and V.r-red. Total DNA delivered was kept constant at 1 μg by supplementing samples (as required) with the corresponding empty control vector. Statistically significant improvements in suppressive activity (*; P < 0.05) at 10 and 50 ng are shown at a finer scale (comparing the single cassette vector to the 2, 3, and 4 cassette vectors). (n = 3) (c) Viral titres of the Infectious virus produced from the 1×, 2×, 3× and 4× all T cassettes vectors, and the corresponding off-target (O') and empty ("e") series (n = 1). (d) Stably integrated HEK293a cell lines for the 1× - 4× T, O' and e cassette vectors were assayed with 300 ng each of T.r and V.r-red, plus 400 ng of the appropriately matched 1 - 4 e cassette vector (to maintain a constant transfection amount of 1 μg) (n = 1). (e) 400 ng each of single shRNA vectors and a series of 1, 2, 3 and 4 cassette vectors made from 4 different shRNAs (T, V, R, and U) were separately assayed with 300 ng each of the 4 corresponding GFP fusion reporters T.r, GFPsVif (V.r), GFPsVpr (R.r) and GFPsVpu (U.r) plus 300 ng of an AsRed1sNef control (N.r-red). (*) There was a statistically significant reduction in the individual suppressive activities of each hairpin relative to their single counterparts (P < 0.01) (n = 1). (f) Net suppressive activities of the same vectors were measured using a HIV-1 production assay by transfecting HEK293a cells with 110 - 130 ng of shRNA vector with 800 ng of pNL4-3 reporter and measuring the impact on p24 production (n = 2).

Activities of different shRNAs from different vectors. (a) The first two test hairpins, Tat 56-29 (T) and Vif 88-29 (V), were expressed from separate plasmid vectors (pS) via the H1 polymerase III promoter. HEK293a cells were transfected with 800 ng of the pSilencer-based (pS) T vector (+ empty) (b), the V vector (+ empty) (c), or the T and V vectors together (d) at mass ratios of 1:7 to 7:1, plus 100 ng each of both target-specific reporter vectors GFPsTat (T.r) and AsRed1sVif (V.r-red). There were 9 data points plotted for each curve, though for clarity open circles are only shown on every 2nd one. The data shown is representative of several replicated experiments (repeated twice, i.e. n = 2).

Activities of multiple-domain, single transcript constructs. (a) Two cluster configurations of R, V and T shRNA domains with (R_V_T₁ and R_V_T₈) and the corresponding single shRNA vectors were assayed using 200 ng of vector plus 400 ng each of R.r, V.r, T.r, and U.r plus N.r-red. The target domains are indicated schematically by: {target}_d. (n = 2) (b) Flow cytometry dot plots from 200 ng of a head-to-tail configuration of the V and T shRNA stem regions (V-T) assayed with 400 ng each of the V.r-red and T.r reporters, showing channel-specific knockdown. (c) The same configuration was assayed with the Vr., T.r and U.r reporters and compared against a similar O'-O'' (control) configuration (similarly assayed) (n > 6). (d) The net activity of the V-T configuration (now expressed from a pL vector) was assayed using 110 - 130 ng of V-T vector and 800 ng of pNL4-3 reporter. (e - h) The component activities of further configurations, T-V, U-R, R-U, R-V-T, T-V-R, V-T₀, V¹⁹-T¹⁹ were similarly assayed using 400 ng of each plus 300 ng of one of the R.r, V.r, T.r, and U.r reporters and 300 ng of N.r-red (as indicated by the appropriately colored boxes) (n >= 1).