Figure 3

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).