• SMARTvector 2.0 Lentiviral shRNA Overview

    New!!!New!!! Have you heard about SMARTchoice RNA?

    As part of the SMARTchoice platform, choose from seven different promoters and two fluorescent reporters to customize SMARTvector 2.0 shRNA constructs.

    Learn more about the SMARTchoice shRNA workflow


    The SMARTvector 2.0 Lentiviral shRNA technology incorporates state-of-the-art design features that enable effective DNA-mediated RNAi

    The shRNA silencing constructs are designed using microRNA scaffold-specific attributes that are integrated into a lentiviral delivery system for efficient processing via the endogenous RNAi pathway.

    SMARTvector 2.0 silencing constructs are delivered at high viral titers for gene silencing experiments suitable for dividing and non-dividing cell types, including difficult-to-transfect cells such as primary cells, neuronal cells and hematopoietic cells.

    Together, these attributes greatly enhance the functionality and specificity of lentiviral-mediated RNAi and reduce the toxicity associated with low titer preparations.

    Superior Dharmacon shRNA Design Algorithm

    • Utilization of a highly processed, proprietary microRNA scaffold
    • Rationally-designed, highly functional gene targeting sequences (Figure 1.)
    • Incorporation of bioinformatics strategies to reduce off-target gene knockdown events

    Ease of Application

    • NEW!!! Tailor your RNAi experiments to specific cells with new promoter and reporter options as part of the SMARTchoice shRNA workflow
    • Engineered for use with a broad range of cells (Figure 2.)
    • Modified with a self-inactivating 3' LTR (SIN vector)
    • shRNA expression linked to detectable TurboGFP/TurboRFP expression
    • Puromycin selection marker for creation of stable cell lines

    Consistent production of high titer (≈108 TU/mL)

    • Provided as transduction-ready concentrated lentiviral particles in DMEM
    • Please note that due to the inherent complexity of high-titer lentiviral particle production, we estimate a 6 to 8 week turnaround time.
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    Highly Functional Gene Knockdown

    Figure 1.
    Highly Functional Gene Knockdown

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    Broad Tropism

    Figure 2.
    Broad Tropism


    SMARTvector 2.0 Lentiviral shRNA Design

    SMARTvector 2.0 gene silencing reagents incorporate design elements critical to the successful delivery and processing of gene targeting sequences.

    Until now, the design of gene targeting sequences for DNA-mediated RNAi has relied predominantly on converting siRNA sequences into hairpins to target genes for knockdown. The developers of the SMARTvector 2.0 Lentiviral shRNA platform found:

    • Highly functional siRNAs do not necessarily provide efficient knockdown when expressed as shRNAs
    • Not all miRNA scaffolds are amenable to the introduction of foreign sequences

    Each element of the vector was experimentally assessed in a systematic series of studies. The results were used to build the most effective and potent lentiviral gene silencing platform available today.

    Our studies led to the incorporation of:

    • Bioinformatic strategies that enhance specificity and reduce off-target silencing
    • An efficiently and correctly processed miRNA expression for specific gene silencing
      • Not all miRNAs are processed with the same efficiency (Figure 1.)
      • Some miRNAs exhibit passenger (star) strand activity that can compromise specificity (Figure 2.)
    • Critical attributes for rational design of the targeting sequence
      • The algorithm selects targeting sequences predicted to be compatible withthe proprietary Thermo Scientific miRNA scaffold
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    Efficiently-Processed microRNA Scaffolds

    Figure 1.
    Efficiently-Processed microRNA Scaffolds

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    Minimal Passanger Strand Activity

    Figure 2.
    Minimal Passenger (Star*) Strand Activity

    SMARTvector: Did You Know?

    Not all promoters perform equally well in all cell types to drive transgene expression. Additionally, the best promoter for your cells may not even be from the same species! Learn more about how the SMARTchoice shRNA workflow can help you tailor your RNAi experiments to your specific cells.

    For a detailed workflow describing the application of SMARTchoice shRNA products, download our Tech Note:

    SMARTchoice shRNA Workflow Tech Note


    SMARTvector 2.0 Lentiviral shRNA Formats

    All SMARTvector 2.0 shRNA products are provided as purified and concentrated packaged lentiviral particles (≈108TU/mL).

    These high, functional titers allow you to perform straightforward RNAi knockdown experiments without the need to create stable cell lines first.

    Ordering Options

    Set of 3 different gene-targeting constructs
    • Ideal for evaluating multiple shRNAs targeting the same gene
    • SMARTchoice shRNA platform offers premium flexibility with the choice of seven promoters (human and mouse CMV, human and mouse eF1α, CAG, PGK, UBC) and two fluorescent reporters (TurboGFP, TurboRFP)
    • 100µL or 200µL sizes at 108 TU/mL (± 20%)
    Individual gene-targeting constructs
    • Ideal for ordering one or two constructs that you have identified as being the best performers
    • SMARTchoice shRNA platform offers premium flexibility with the choice of seven promoters (human and mouse CMV, human and mouse eF1α, CAG, PGK, UBC) and two fluorescent reporters (TurboGFP, TurboRFP)
    • 100µL or 200µL sizes at 108 TU/mL (± 20%)
    Matched control particles using the SMARTchoice shRNA platform
    • Select controls with identical promoter-reporter combinations to gene-specific SMARTvector 2.0 viral particles
    • 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

    At Thermo Fisher Scientific, 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 viral particle production and the quality control procedures, turn-around time is estimated to be 6 to 8 weeks.

    SMARTvector 2.0 Lentiviral shRNA Related Products

    Ancillary products for the SMARTvector experimental workflow include:

    SMARTchoice shRNA Promoter Selection Plate
    96-well plate containing duplicate serial dilution series of SMARTvector 2.0 Lentiviral shRNA Non-targeting Control Particles constructed with seven different promoters and expressing TurboGFP. Evaluate the activity of multiple promoters simultaneously by assessing fluorescence intensity in your specific cells of interest.
    SMARTvector Positive Control Particles
    Packaged lentiviral particles used to confirm transduction efficiency by assessing TurboGFP expression and evaluate specific and potent silencing of a non-essential, abundantly expressed housekeeping gene or expression of a fluorescent protein from a reporter plasmid
    SMARTvector Negative Control Particles
    Packaged lentiviral particles used to confirm silencing specificity of gene-targeting SMARTvector 2.0 Lentiviral shRNA Particles and distinguish sequence-specific silencing from non-specific effects
    Solaris qPCR Assays
    Solaris qPCR Gene Expression Assays are pre-designed, gene specific probe and primer pairs that utilize minor groove binder (MGB) and SuperBase technology to deliver repeatable, sensitive and gene specific quantification. Optimal results are obtained when used as a complete system with Solaris qPCR Master Mix.

    Additional Related Thermo Scientific Products

    PCR Plastics
    Premium molecular grade plastics designed, manufactured and tested specifically for PCR performance.
    PCR Reagents
    A portfolio of products forend-point PCR that includes high quality reagents for any PCR application.
    Cellomics HCS Reagent Kits
    For cell-based screening and high-content imaging of knockdown phenotypes
    Pierce Protein Research Products
    For analysis of gene silencing at the protein level

    SMARTvector 2.0 Rational Design Provides Highly Functional Gene Knockdown

    shrna-smartvector-design-knockdown-figure

    Figure 1. Performance of the SMARTvector 2.0 rational design algorithm. The top five scored sequences targeting EGFR, MapK1, CDC2 and APD were tested for the ability to knockdown dual-luciferase reporter constructs containing complementary target sequences. Greater than 75% silencing was achieved with 18 of the 20 SMARTvector 2.0 constructs.

    The SMARTvector 2.0 rational design algorithm incorporates multiple determinants including:

    • Thermodynamic profiles for strand bias
    • Global and region-specific GC content
    • Secondary structures essential for correct processing
    • Position-dependent nucleotide preferences
    • Seed-complement frequency filters

    VSVg Envelope Provides Broad Tropism

    SMARTvector Broad Tropism

    Figure 2. Ten different cell lines were transduced with Dharmacon SMARTvector 2.0 Lentiviral shRNA Particles targeting either RHOA, GAPD or RAC1 (as indicated) at three MOIs. mRNA knockdown was assessed 72 hours post-transduction using QuantiGene branched DNA assay (Panomics, Inc.) and normalized toSMARTvector 2.0Non-targetingControl. In most cases, 75% or greater gene knockdown was achieved in the absence of puromycin selection.

    Rational design, broad tropism, and GFP reporter expression facilitate transduction optimization that leads to efficient gene knockdown in cells refractory to lipid-mediated delivery.

    Highly functional miRNA scaffolds (>80% knockdown) were chosen for SMARTvector 2.0 lentiviral shRNA development

    Highly Functional miRNA scaffolds

    Figure 1. Incorporation of highly-functional and appropriately-processed miRNA scaffolds into lentiviral silencing constructs greatly enhances the functionality of DNA-based RNAi silencing.

    • Ten separate endogenous human miRNAs were tested in HeLa cells for the ability to silence their respective targets. Each miRNA exhibits a distinctive level of functionality.
    • Several of the constructs (e.g., miR-486 and -526a) exhibited poor levels of activity, silencing their respective reporter constructs less than 20% while native miR-30 and -374 scaffolds provide moderate levels of gene silencing (~50-70%, see above).
    • Four scaffolds (SMARTvector A-D) were efficiently processed and silenced their respective targets by 80 to 90%. These highly functional miRNAs represent a significant improvement over currently available miRNA-based scaffolds used in RNAi expression platforms.

    SMARTvector 2.0 miRNA Scaffolds Exhibit Minimal Passenger (Star*) Strand Activity

    SMARTvector 2.0 miRNA scaffolds strand acticity

    Figure 2. Preferred miRNA scaffolds have minimal passenger strand off-target effects. The Thermo Fisher Scientific team investigated the passenger strand activity of several of the scaffolds tested in the primary screen. The moderately functional miRNA-f and the highly functional miRNA-e exhibited extensive passenger strand activity and were not considered for further development. In contrast, miRNA-a and miRNA-c scaffolds showed preferential mature strand entry.

    miRNA scaffolds having potent mature strand activity with minimal passenger strand function were selected for SMARTvector shRNA construct development.