Optimal shRNA expression for high-confidence functional analysis results
Our most advanced shRNA platform, SMARTvector lentiviral shRNAs utilize the flexibility of promoter and reporter options 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.
- Innovative vector design for robust shRNA expression from SMARTvector Lentiviral shRNAs
- SMARTchoice options give you a choice of promoter* and reporter (or no reporter!) for optimal performance in your experiment.
- Rationally designed shRNAs for maximum knockdown and minimal off-targeting
Choose SMARTvector inducible lentiviral shRNA for tight regulation of expression from the Tet-On® 3G transactivator protein
*Some promoter options may only be available as custom reagents or upon request.
Each SMARTvector lentiviral shRNA library contains
- ≥ 5 x 108 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 containing construct sequences, target gene IDs, and counts per millions of mapped reads
SMARTvector 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 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
Before ordering, download these invaluable tools to carefully plan your pooled lentiviral shRNA screen and calculate the amounts of components required:
- SMARTvector & shMIMIC lentiviral pooled libraries technical manual
- Pooled Lentiviral Screening Laboratory Protocols & Calculation Tracking Worksheet
Reagents recommended in our validated protocol:
- Vector-matched SMARTvector 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
SMARTvector lentiviral shRNA pooled library coverage*:
|# 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|
|Cell cycle regulation||**|
|DNA damage response||**|
The SMARTvector 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. Reagents 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 reagents 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.
- Ž. Strezoska, A. Licon, Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens. PLoS One 7, e42341 (2012).
Elements of the SMARTvector Lentiviral shRNA Backbone
|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|
|PRRE||Rev Response Element enhances titer by increasing packaging efficiency of full-length viral genomes|
|tGFP or tRFP||TurboGFP or TurboRFP reporter for visual tracking expression upon doxycycline induction|
|None||No-reporter option for use in applications where fluorescence is not required or desired|
|IRES||Internal Ribosomal Entry Site allows expression of fluorescent marker and puromycin resistance in a single transcript|
|PuroR||Puromycin resistance permits antibioticselective pressure and propagation of stable integrants|
|SMARTvector universal scaffold||Optimized proprietary scaffold in which mature microRNA sequence is embedded|
|WPRE||Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells|
|3' SIN LTR||3' Self-inactivating Long Terminal Repeat for increased Lentiviral safety|
SMARTvector Pooled 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.
Strength of promoter activity varies from one biological context to another and can affect experimental outcomes.
Promoter activity varies across several human and rodent cell lines. Cells were plated at a density of 50,000 cells per well in a 24-well plate and transduced at MOI = 15 with SMARTvector Empty Vector Control Particles expressing TurboGFP. Promoter activity was assessed at 72 hours post-transduction by the fluorescence intensity of TurboGFP.