Obtain Efficient Delivery and Permanent Knock Down SBI’s shRNA and siRNA Lentiviral Cloning and Expression System enables you to set up stable heritable gene silencing systems. You can easily and efficietly transduce dividing and quiescent mammalian cells—including difficult-to-transfect cells such as primary, stem, neuronal, and endothelial. In addition, our RNAi vectors can be used to generate gene-specific knockdown animals, such as transgenic mice.
Analyze the Specific Effects of Target Genes Lentiviral transduction eliminates the side effects produced by harsh, disruptive, and inefficient transfection protocols using chemically synthesized siRNA. The use of highly efficient and non-stressful lentiviral transduction greatly increases the likelihood that any observed phenotypic effects result from target-gene silencing.
Thoroughly Understand Target Gene Function The Lentivector system provides a convenient and effective approach to create stable cell lines or transgenic animals since it efficiently integrates the siRNA expression construct of your choice into genomic DNA. Assays with cell lines or transgenic animals that permanently and heritably maintain a gene specific knock down phenotype enable you to more thoroughly analyze the specific effects resulting from silencing your target gene.
Unique, Highly Effective System SBI’s FIV and HIV-based Cloning and Expression Lentivectors are derived from self-inactivating lentiviral vectors. This unique system offers a highly effective and very safe approach for introducing and expressing any siRNA sequence in nearly any mammalian cell system.
Single or Double Promoter Formats The FIV and HIV-based shRNA lentivectors are both available in a single-promoter format, and they contain the lentiviral genetic elements for expression of high levels of siRNA in the target cell. In addition, we offer novel double-promoter FIV-based lentivectors which feature opposing promoters that directly generate double-stranded siRNA.
Complete Lentivector Packaging System In order to stably transduce cells with SBI’s lentivectors, use SBI’s pPACK Lentivector Packaging System together with our 293TN cell line. The packaging system provides all the necessary coat proteins to manufacture pseudoviral particles for delivery of the siRNA expression construct into any mammalian cells or model organisms.
Stably Express Short-Hairpin RNA (shRNA)
Potent RNA interference using shRNAs
The shRNA template sequence is cloned into the unique BamHI/EcoRI sites. The H1 (or U6) RNA polymerase III promoter expresses a single oligonucleotide template that codes for both the sense and antisense portions of the siRNA molecule. The expressed shRNA folds into a stem-loop-stem structure that is processed by the DICER enzyme to produce an active siRNA molecule.
After transduction, positive clones can be isolated by sorting with a flow cytometer, selecting for puromycin resistance, or by staining cells with H2Kk-FITC antibodies. Vectors are also available pre-packaged in order to compare efficiencies of different promoters (see Lentiviral Products).
The pSIH and pSIF shRNA cloning vectors are available in a variety of formats. Choose from a CMV, EF1, PGK, or CMV/Ferritin promoter driving expression of the copGFP reporter or Puromycin resistance gene. For shRNA expression, choose from the H1 or U6 promoter. The pGreenPuro™ shRNA vector enables both GFP monitoring and Puromycin selection.
shRNA expression vectors for GFP sorting and Puromycin selection
HEK-293 cells were transfected with either pSIH1-H1-copGFP or pGreenPuro™ shRNA expression vector, and puromycin (8 – 50 ug/ml final concentration) was then added to the cells 24 hours after transfection. The pictures were taken 24 hour after the initiation of the puromycin treatment.
shRNA expression vectors also available in Clone-it™ format
shRNA expression vectors are also available in Clone-it ligase free cloning systems (cat.# LF523A-1, LF524A-1). The Ligase-free Cloning Site (LCS) replaces the BamHI and EcoRI cloning sites in the pSIH-H1 type lentivectors. More Clone-it™ product information.
Express siRNA Directly
Express natural double-stranded siRNA constructs (as opposed to hairpin-type siRNA). The expression cassette is flanked by opposing H1 and U6 RNA Polymerase III promoters and T5 terminator sequences. Double-stranded siRNA templates are easier and less expensive to synthesize, easier to clone, and more stable during propagation. These features make this vector easy to use, cost-effective, but the silencing efficiency achieved with these types of vectors is usually less than with single-promoter vectors. Successfully cloned siRNA template oligonucleotides are transcribed in both directions.
