SMARTvector Inducible Lentiviral shRNA Pooled Libraries

Inducible shRNA expression for regulatable and controlled functional analysis screens


SMARTvector Inducible Lentiviral shRNA Pooled Libraries
Combine the advantages of rationally designed shRNAs and the flexibiltiy of selectable promoters and reporters with the tight control of the Tet-On 3G tetracycline-inducible transactivator for unmatched shRNA expression

Optimal shRNA expression for high-confidence functional analysis results

Our most advanced shRNA platform, SMARTvector Inducible Lentiviral shRNAs utilize the flexibility of promoter and reporter options and the tight regulation of expression from the Tet-On® 3G transactivator protein coupled with our most advanced rational design algorithm to target the complete genome for human, mouse, and rat protein-coding and long non-coding genes.

Pooled lentiviral libraries offer an efficient and cost-effective method for screening large numbers of genes without automation. Library construction, pooling techniques, and high-throughput sequencing-compatible screening workflows have been experimentally validated to ensure reproducibility and accurate hit identification. Learn about optimal library design and the pooled screening workflow on our Pooled Lentiviral Screening Libraries page.

Highlights:

Choose SMARTvector Lentiviral shRNA Pooled Libraries for constitutive shRNA expression.

Each SMARTvector Inducible Lentiviral shRNA Library contains:

  • ≥ 1 x 107 TU/mL (+/- 20 %) lentiviral particles provided in pre-aliquoted tubes
    • 8 x 25 µL (200 µL total) for libraries ≤ 5000 constructs
    • 16 x 25 µL (400 µL total) for libraries > 5000 constructs
  • 100 Non-targeting control constructs
  • 272 constructs targeting 34 human or mouse protein coding genes as gene-specific controls (8 shRNA per gene); includes common reference genes (PPIB, GAPD, Actin, Lamin) and viability-related genes.
  • A data file including construct sequences, target gene IDs, and counts per millions of mapped reads

SMARTvector Inducible Lentiviral shRNA libraries are available for defined sub-libraries of gene families up to the entire genome for human and mouse; rat libraries and custom SMARTvector Inducible Lentiviral shRNA collections are available upon request.

  • Request a custom collection of human, mouse, or rat gene-targeting shRNAs
  • Available in pool sizes between 50 to 10,000 constructs
  • Pools are provided as purified, concentrated lentiviral particles
  • ≥ 5 x 108 TU/mL for constitutive vectors and ≥ 1 x 107 TU/mL for inducible vectors

Request pricing for a custom pooled library

Products recommended in our validated protocol:

  • Vector-matched SMARTvector Inducible Non-targeting control for transduction optimization
  • SMARTvector Indexing PCR and Sequencing Primer Kits (A & B) with 12 unique indexing primers, optimized and experimentally validated for:
    • Efficient PCR amplification of genomic DNA with minimal bias
    • High-throughput multiplexed Next Gen Sequencing for hit identification

Before ordering, download these invaluable tools to carefully plan your pooled lentiviral shRNA screen and calculate the amounts of components required:

SMARTvector Lentiviral shRNA pooled library coverage*:

*Annotations are based on RefSeq v65; coverage and shRNA counts are subject to change without notice
** Clone and gene counts available by request
Collection Human   Mouse  
  # targeted genes Number pools and average shRNA per pool # targeted genes Number pools and average shRNA per pool
Genome 19241 16 pools of 9513 shRNA 21745 18 pools of 9502 shRNA
Druggable Genome 7341 6 pools of 9735 shRNA 9723 8 pools of 9670 shRNA
GPCR 377 1 pool of 3012 shRNA 494 1 pool of 3909 shRNA
Ion Channel 340 1 pool of 2709 shRNA 332 1 pool of 2646 shRNA
Phosphatase 245 1 pool of 1948 shRNA 268 1 pool of 2141 shRNA
Protease 466 1 pool of 3702 shRNA 529 1 pool of 4206 shRNA
Protein Kinase 702 1 pool of 5602 shRNA 697 1 pool of 5558 shRNA
Ubiquitin Conjugation 557 1 pool of 4431 shRNA 511 1 pool of 4069 shRNA
Apoptosis **
Cell Cycle Regulation **
De-ubiquitinating Enzymes **
Membrane Trafficking **
DNA Damage Response **
Epigenetics **
Nuclear Receptor **
Transcription Factors **

 

Important Notice

The SMARTvector Inducible Lentiviral shRNA Pooled Libraries are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where the containment measures stated below and in applicable laws and regulations are met. Products may not be used for diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any purpose or to animals for therapeutic purposes. SMARTvector Lentiviral shRNA Pooled Libraries are provided as lentiviral particles are replication-incompetent, self-inactivating (SIN) and non-pathogenic (do not cause infectious human disease).

Any investigator who purchases lentiviral particle products is responsible for consulting with their institution's health and biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully responsible for obtaining the required permissions for research using and the acceptance of replication-incompetent SIN lentiviral vectors and replication-defective lentiviral particles into their local jurisdiction and institution.

  
HazardousNo
Shipping ConditionDry Ice
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-80 C
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Elements of the SMARTvector Inducible shRNA Lentiviral Backbone

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Vector Element Utility
5' LTR 5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genome
Ψ Psi packaging sequence allows viral genome packaging using lentiviral packaging systems
PTRE3G Inducible promoter with Tetracycline Response Element is activated by the Tet-On® 3G protein in the presence of doxycycline
tGFP or tRFP TurboGFP or TurboRFP reporter for visual tracking expression upon doxycycline induction
SMARTvector universal scaffold Optimized proprietary scaffold based on native primary microRNA in which gene-targeting sequence is embedded
PuroR Puromycin resistance permits antibiotic selection of transduced cells
2a Self-cleaving peptide enables the expression of both PuroR and Tet-On® 3G transactivator from a single RNA pol II promoter
Tet-On® 3G Encodes the doxycycline-regulated transactivator protein, which binds to PTRE3G promoter only in the presence of doxycycline
WPRE Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells
3' SIN LTR 3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles


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An Optimized Inducible Gene Silencing System in a Single Vector

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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.


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Pooled SMARTvector Screening Workflow

Pooled-shRNA-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.


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SMARTvector Inducible Lentiviral shRNA promoter activities differ across cell types

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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.


HUVEC

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

HUVEC

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.


essential gene U2OS

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

essential gene U2OS

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.


References

  1. Tet-inducible expression system references:

    Loewr, Heinz N, et al., Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol. 10, 81 (2010).

    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).

  2. Ž. Strezoska, A. Licon, Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens. PLoS One 7, e42341 (2012).