Characteristics of the pSLIK single vector system for conditional RNAi. (a) Schematics of the pSLIK-Venus and pSLIK-Neo vector platforms. Validated miR-shRNAs in the pEN_TmiRc2 vector are subcloned by site-specific recombination to either pSLIK vector. (b) Schematic showing predicted constitutive (black) and DOX-induced (green) transcripts from the pSLIK lentivirus. (c) FACS analysis of Venus expression in pSLIK-Venus transduced MEFs in the absence (−) and presence (+) of DOX (1 μg/ml). The presence of DOX induces a 10-fold increase in the Venus mean fluorescence intensity. (d) DOX-dependent increase in rtTA3 and Neo transcript expression in pSLIK-Neo transduced RAW264.7 cells. Neo selection with G418 was withdrawn from cells during DOX induction. mRNA levels were assessed by quantitative RT-PCR and normalized to the expression level in the absence of DOX (1 μg/ml). (e) Kinetics of conditional Gα12 knockdown in MEFs. Western blot analysis of Gα12 protein levels in pSLIK-Venus-Gα12 transduced MEFs cultured in the presence of 1 μg/ml DOX for 8 days (DOX addition). (f) Gα12 knockdown is readily reversed during continued culture for 8 days in the absence of DOX. (g) DOX dose–response in pSLIK transduced MEFs. Potent Gα12 target protein knockdown is induced by 100 ng/ml DOX.

Multigene knockdown using pSLIK. (a) Specificity of miR-shRNAs against Gα12 and Gα13. MEFs transduced with pSLIK lentiviruses expressing Gα12 and Gα13 miR-shRNAs mediate specific knockdown of their target proteins in the presence of DOX (1 μg/ml). (b) Schematic showing creation of pSLIK lentivirus encoding tandem miR-shRNAs. miR-shRNAs are concatenated in the pEN_TmiRc2 entry vector by directional cloning (S, SpeI; X, XbaI; P, PstI), then subcloned to pSLIK-Venus by site-specific recombination. (c–e) Assessment of Gα12 (c) and Gα13 (d) mRNA and protein (e) levels in MEF cell lines transduced with control, Gα12, Gα13, and Gα12 plus Gα13 miR-shRNA-expressing pSLIK viruses. RNA and protein were harvested from cells cultured in the absence or presence of DOX (1 μg/ml) for 5 days. mRNA levels were assessed by quantitative RT-PCR and normalized to the expression level in untreated control cells. (f) LPA induced SRE-dependent transcription in MEF cell lines transduced with control, Gα12, Gα13, and Gα12 plus Gα13 miR-shRNA-expressing pSLIK viruses. Cells transfected with SRE-Luc and pCSK-lacZ were stimulated with 5 μM LPA for 7 h. Luciferase activity in cell lysates was assessed from cells cultured in the absence or presence of DOX (1 μg/ml) for 5 days and was normalized to the level of cotransfected β-galactosidase.

pSLIK-based conditional knockdown of Gβ2 in RAW264.7 macrophages demonstrates Gβ2 selectivity in C5a-mediated Ca²⁺ response. (a) Western blot analysis of RAW cells transduced with a pSLIK-Neo-Gβ2 lentivirus. (b) Time course of target protein knockdown in response to 1 μg/ml DOX for 7 days and recovery kinetics through 7 days after DOX removal. (c) Ca²⁺ response profiles in pSLIK-Neo-Gβ2 transduced RAW cells treated with C5a (10 nM). Cells were cultured in the absence or presence of 1 μg/ml DOX for 2 days before assay. (d) Effect of DOX-dependent Gβ2 knockdown on the C5a-induced Ca²⁺ peak in pSLIK-Neo-Gβ2 transduced RAW cells. Data were averaged from cells induced with 1 μg/ml DOX for 2 and 4 days and normalized to the peak offset in cells cultured in the absence of DOX.

Coupling of conditional miR-shRNA and transgene expression from pSLIK in RAW264.7 macrophages. (a) Schematic showing insertion of a GFP transgene in a pSLIK-Neo lentivirus encoding the Gβ2 miR-shRNA. (b) Western blot analysis demonstrates that conditional GFP expression is tightly correlated with conditional Gβ2 knockdown. Upon DOX removal, Gβ2 expression is restored. GFP expression is minimal after 2 days and is completely absent after 4 days.