Previously added items:
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 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.
Highly Functional Gene Knockdown
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:
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:
Efficiently-Processed microRNA Scaffolds
Minimal Passenger (Star*) Strand Activity
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
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.
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.
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:
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.
Figure 1. Incorporation of highly-functional and appropriately-processed miRNA scaffolds into lentiviral silencing constructs greatly enhances the functionality of DNA-based RNAi silencing.
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.