pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit

Using the pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit, you can:

•Assess the potency of any RNAi knockdown reagent using a fast, sensitive, and cost-effective β-gal reporter assay
•Identify the best knockdown reagent for your RNAi experiments without knowing protein function, western blotting, or qRT-PCR

How it Works: The BLOCK-iT™ RNAi Target Screening system method uses a β-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth RNAi™ siRNA, siRNA, dicer pools, or shRNA-containing plasmid). To identify the most effective knockdown reagent for your target, simply clone your target gene into the pSCREEN-iT™ /lacZ-DEST Gateway™ vector. Co-transfect the resulting construct with the knockdown reagent being tested. Expressed genes will be fused to β-galactosidase, enabling you to use β-galactosidase levels to measure the amount of RNAi-induced degradation of the target gene (Figure 1). The more effective any specific knockdown reagent is at mediating RNAi, the more degradation of the fusion mRNA will occur, lowering β-gal activity (Figure 2).

The BLOCK-iT™ RNAi Target Screening System includes the pSCREEN-iT™/lacZ-DEST Gateway™ vector for generating a construct containing a lacZ/gene of interest fusion. This vector features:

•attR sites immediately following the lacZ ORF for fast and efficient recombination with any attL-flanked Gateway™ entry vector to generate the lacZ/target gene fusions
•The CMV promoter for strong expression of the lacZ/target gene fusion, leading to increased assay sensitivity and better discrimination between knockdown reagents of varied effectiveness
•Positional cloning to fuse a gene or fragment to the 3´ end of the lacZ gene so that β-galactosidase will be expressed even if the target fragment being tested contains stop codons

In addition, the pSCREEN-iT™/lacZ-DEST vector is fully compatible with the Ultimate™ ORF Clone Collection. This makes a wide selection of potential target genes readily available for generating lacZ fusions.