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).