A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression

Abstract

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Fig. 1.

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 Neo selection with G418 was withdrawn from cells during DOX induction.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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.