SMARTvector Inducible Lentiviral shRNA
The market standard for inducible RNAi
Combining all of the advantages of the SMARTvector shRNA design innovations with the recently developed Tet-On 3G tetracycline-inducible expression system and the flexibility of SMARTchoice promoter and reporter options, this novel single-vector regulatable RNAi system permits the rapid development of stable cells with tightly controlled shRNA expression.
SMARTvector Inducible Lentiviral shRNAs are designed using our most advanced microRNA-based rational shRNA-specific design algorithm resulting in highly potent and specific targeting sequences. Choose from four promoters (mouse CMV, human and mouse EF1 alpha, and PGK) and two fluorescent reporters (TurboGFP and TurboRFP) to tailor SMARTvector Inducible Lentiviral shRNAs for your specific cells of interest and your experimental requirements. Up to ten shRNA silencing constructs are predesigned for every gene in human, mouse and rat providing broad coverage across the entire genome.
The minimal leakiness and potent activation of the Tet-On 3G promoter ensures near wild-type expression of the target gene in the absence of doxycycline and robust gene silencing upon exposure to doxycycline. When induced, SMARTvector Inducible Lentiviral shRNA is incredibly potent, resulting in highly efficient knockdown even at very low multiplicities of infection (MOI).
Unsure which format you need? Learn more about our SMARTchoice configuration platform.
SMARTvector Inducible Lentiviral shRNA vectors possess the following enhanced features for robust, regulatable, and reproducible shRNA-based gene silencing:
- Target any gene in human, mouse and rat, and tailor experiments to specific cells with multiple promoter options
- Guaranteed silencing (see Guarantee tab)
- Designed using microRNA scaffold-specific attributes for highly efficient processing via the endogenous RNAi pathway
- Tight control of shRNA and reporter gene expression utilizing the latest generation Tet-inducible expression technology, the Tet-On® 3G Inducible System
- Available as glycerol stocks and as high-titer purified, concentrated lentiviral particles at 1 x 107 TU/mL, ± 20%; functional titers determined by flow cytometric analysis of GFP-positive, transduced HEK293T cells
- Suitable for dividing and non-dividing cell types, including difficult-to-transfect cells such as primary, neuronal and stem cells
Together, these attributes greatly enhance the functionality and specificity of lentiviral-mediated RNAi and reduce the toxicity associated with low titer preparations (supernatant).
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 third-generation Tet-inducible system significantly improved and optimized for minimal basal expression (lowest leakiness) and potent activation upon induction (see References tab). The Tet-On® 3G Inducible System permits tightly controlled shRNA expression and study of gene function in vivo and in vitro with unprecedented precision.
Flexibility to choose your SMARTvector Inducible Lentiviral shRNA vector attributes
Inducible control of the SMARTvector shRNA and the reporter gene are conferred by the Tet 3G promoter (PTRE3G), while the expression of the Tet-On® 3G transactivator protein is under the control of a constitutive RNA pol II promoter. The choice of the constitutive RNA pol II promoter is critical to ensure robust production of PuroR and Tet-On® 3G transactivator protein, thereby allowing antibiotic selection of transduced cells and rigorously controlled expression of the shRNA. Promoter activity varies significantly across different cell lines which, in turn, can influence the efficacy and potency of shRNA-mediated gene silencing. The SMARTchoice inducible vector customization options include four constitutive promoter and two reporter options, thus maximizing the opportunity for RNAi experimental success.
Superior Dharmacon SMARTvector Lentiviral shRNA rational design algorithm for potent knockdown at single-copy integration
- Utilization of a highly processed, patented microRNA scaffold
- Preferential mature strand RISC-loading and activity with minimal processing and activity of passenger (non-guide) strand
- Rationally designed, highly functional gene targeting sequences
- Algorithm trained on more than 500 endogenous mRNA data points for functional knockdown testing
- Incorporation of bioinformatics strategies to reduce off-target gene knockdown events
- Selection of shRNA designs with low microRNA seed-complement frequency
- Additional filtering of shRNA designs for known microRNA and toxicity nucleotide motifs
- Protein coding genes are targeted in the ORF and 3’UTR; lncRNAs are targeted throughout the entire transcript length
Achieve unprecedented levels of sensitivity and gene silencing control for your most sensitive biological applications
The ability to control when and to what degree a gene is silenced has proven invaluable to the task of elucidating gene function in vitro and in vivo, under biologically relevant physiological and disease-associated conditions. Regulatable RNAi systems are particularly useful for functional analysis of genes that, when silenced, may cause deleterious effects or irreversible changes in the cellular state and dynamics. The SMARTvector Inducible Lentiviral shRNA platform facilitates many potential applications, including:
IN VITRO applications such as
- Studying gene function
- Modeling pathways and networks
- Manipulating fate of adult/embryonic cells
- Engineering cell models of disease
- Validating drug targets and mechanism of action (MOA)
EX VIVO applications such as
- Probing tumor phenotypes
- Manipulating fate of adult/embryonic cells
IN VIVO applications such as
- Modeling progressive diseases
- Developing transgenic models
Lentiviral particle formats
At Dharmacon, we are committed to delivering high quality lentiviral particle preparations. As such, we clone, package and concentrate each construct with strict QC controls. Due to the inherent complexity of lentiviral particle production and the quality control procedures, turnaround time is estimated to be 4 to 6 weeks. We offer the following formats:
Set of 3 different gene-targeting constructs
- Ideal for evaluating multiple shRNAs targeting the same gene and ensuring mRNA knockdown in your specific cells
- 100 µL (4 x 25 µL) or 200 µL (8 x 25 µL) total amounts at 1 x 107 TU/mL, ± 20%, functional titers determined by flow cytometric analysis of GFP-positive, transduced HEK293T cells
Individual gene-targeting constructs
- Ideal for ordering one or two constructs that you have identified as being the best performers
- 100 µL (4 x 25 µL) or 200 µL (8 x 25 µL) total amounts at 1 x 107 TU/mL, functional titers determined by flow cytometric analysis of GFP-positive, transduced HEK293T cells
SMARTvector Inducible Lentiviral Positive and Negative Controls for shRNA and microRNA experiments
- Ideal for optimizing transduction and assay conditions prior to gene-specific experimentation
- Negative control particles allow for distinguishing sequence-specific silencing from non-specific effects
- Positive control particles allow for confirmation of gene silencing using confirmed shRNA constructs targeting a housekeeping gene
- 50 µL (2 x 25 µL) at 1 x 107 TU/mL, ± 20%, functional titers determined by flow cytometric analysis of GFP-positive, transduced HEK293T cells
SMARTvector Inducible Lentiviral shRNA functional guarantee
When you purchase a minimum of three SMARTvector Inducible Lentiviral shRNAs to the same protein-coding gene target using the optimal SMARTchoice promoter and doxycycline dosage for your cell type, at least one of the shRNA constructs will reduce target mRNA levels by 70% or more when used with the vector matched non-targeting control and GAPD or PPIB positive control. Optimal promoters should be determined with the SMARTchoice Inducible Non-targeting Control 4-pack. Optimal doxycycline conditions should be determined as described in the SMARTvector Inducible Lentiviral shRNA Technical Manual. mRNA levels should be measured with RT-qPCR or similar quantitative mRNA analysis no earlier than 72 hours post-doxycycline induction. The guarantee only applies to catalog numbers V3S*xxxx and does not apply to any SMARTvector Inducible Lentiviral shRNAs targeting lncRNA transcripts.
- Inducible expression of positive and negative controls with your choice of four promoters and two reporters.
Elements of the SMARTvector Inducible shRNA Lentiviral 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|
|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|
An Optimized Inducible Gene Silencing System in a Single Vector
Induction of SMARTvector Inducible Lentiviral shRNA expression in a cancer cell line is robust and tightly controllable in a doxycycline dose-dependent manner
Induction of SMARTvector Inducible Lentiviral shRNA expression is temporally controlled and results in highly efficient gene silencing at very low MOI
SMARTvector Inducible Lentiviral shRNAs permit tunable knockdown of specific target genes in mouse cells with all four promoter options
SMARTvector Inducible Lentiviral shRNAs permit tunable knockdown of specific target genes in human primary cells with all four promoter options.
SMARTvector Inducible Lentiviral shRNAs enable tightly controlled knockdown of essential genes in a time- and doxycycline dose-dependent manner
SMARTvector Inducible Lentiviral shRNA promoter activities differ across cell types
Functionality of endogenous microRNAs are different
Functionality of mature and passenger strands of endogenous microRNAs are different
microRNA-adapted shRNAs compared to simple hairpin shRNAs
Tet -inducible expression system references:
- Loew R, 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).