Elements of the SMARTvector Inducible shRNA Lentiviral Backbone

An Optimized Inducible Gene Silencing System in a Single Vector

The SMARTvector Inducible Lentiviral shRNA vectors incorporate the Tet-On® 3G bipartite induction system a 3rd-generation Tet-inducible system significantly improved and optimized for minimal basal expression (lowest leakiness) and potent activation upon induction (Zhou X, et al., Gene Therapy 13, 1382 (2006) and Loew R, et al., BMC Biotechnol. 10, 81 (2010)). The Tet-On® 3G Inducible System permits tightly controlled shRNA expression and study of gene function in vivo and in vitro with unprecedented precision.

Pooled SMARTvector Screening Workflow

Assay Development and Optimization: Establish optimal experimental conditions, including those for a) lentiviral transduction and b) screening parameters, such as selective pressure and time between collection of reference and experimental samples.Primary Screen: A stable population of cells expressing single integrands of constructs are created by transducing lentiviral pools at low MOIs. Transduced cells are then split into reference and experimental populations for application of a selective pressure that induces the phenotype of interest. Genomic DNA (gDNA) is then isolated from reference and experimental populations of transduced cells. Illumina-adapted primers and Phusion Hot-Start II High Fidelity DNA Polymerase are used to PCR amplify integrated construct sequences and add Illumina flow-cell binding sequences. The resulting amplicons are run on Illumina platform sequencers, using the sequencing primers provided.Hit Identification and Follow-up: Construct sequences are identified in reference and experimental libraries. Constructs that are enriched or depleted during the screen are identified as hits, and the genes that they target are identified. Hits can be confirmed and studied further using individual constructs that can be ordered from the Dharmacon catalog collection.

SMARTvector Inducible Lentiviral shRNA promoter activities differ across cell types

SMARTvector Inducible Lentiviral shRNA Non-targeting Controls were used to transduce the indicated cell types at MOI = 0.3. 24 h after transduction, expression of the non-targeting shRNA and PuroR was induced with 1 µg/mL doxycycline. After 48 h of culture in the presence of doxycycline, cells were stained with Hoescht-33342 and nuclei (blue) and TurboGFP (green) were imaged. While ~30% of the cells in the field have been transduced, some images may appear to contain fewer than 30% TurboGFP-positive cells due to low TurboGFP expression, indicative of low constitutive promoter activity, in a particular cell type.

SMARTvector Inducible Lentiviral shRNAs permit tunable knockdown of specific target genes in human primary cells with all four promoter options.

HUVEC (primary human umbilical vein endothelial) cells were transduced at MOI = 0.2 with the indicated SMARTvector Inducible Lentiviral shRNA vectors carrying a non-targeting control shRNA (NTC) or shRNAs directed against RHOA or GAPDH. Cells were selected with 1.5 µg/mL puromycin for 72 hours. shRNA expression was then induced with 0, 0.01, 0.1, 1.0 and 10 µg/mL doxycycline, respectively, and mRNA was harvested 72 hours after dox-induction. Target gene silencing was measured by RT-qPCR relative to the PPIB reference gene. Gene silencing is expressed relative to the vector- and doxycycline dosage-matched NTC cell population.

SMARTvector Inducible Lentiviral shRNAs enable tightly controlled knockdown of essential genes in a time- and doxycycline dose-dependent manner

U2OS cells were transduced at MOI = 0.1 with SMARTvector Inducible Lentiviral mCMV vectors carrying either a non-targeting control shRNA (NTC) or an shRNA directed against the ubiquitin B (UBB) gene. Cells were selected for 3 days with 1.5 µg/mL puromycin. After selection, cells were seeded at 2000 cells per/well in 96-well plates. 24 hours later (Day 1), shRNA expression was induced with the indicated dose of doxycycline, and cell number was then measured each day with the Cell Titer-Glo® assay (Promega). Each data point represents the mean and standard deviation of six independent wells. B. On Day 6, following 5 days of exposure to doxycycline, the cells were stained with Hoescht 33342 and cell nuclei (blue) and TurboGFP (green) were imaged on the ArrayScan™ VTI HCS Reader. C. Cell density, defined as number of nuclei per field was computed for 18 fields per condition.

Tet-inducible expression system references:

1. Loewr, Heinz N, et al., Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol. 10, 81 (2010).
2. Zhou X, Vink M, et al., Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther. 13(19), 1382-1390 (2006).
3. Ž. Strezoska, A. Licon, Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens. PLoS One 7, e42341 (2012).