BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP (Invitrogen™)

The BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP combines Invitrogen's BLOCK-iT™ Pol II miR RNAi and ViraPower™ Lentiviral technologies to facilitate creation of a replication-incompetent lentivirus that delivers a microRNA (miRNA) sequence of interest to dividing or non-dividing mammalian cells for RNA interference (RNAi) analysis thus broadening the potential RNAi applications beyond those of other traditional retroviral systems (Naldini, 1998). The BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP includes: o A BLOCK-iT™ Pol II miR RNAi Expression Vector Kit for production of an expression clone containing a double-stranded oligonucleotide encoding a pre-miRNA sequence for expression in mammalian cells. The BLOCK-iT™ Pol II miR RNAi Expression Vector (pcDNA™6.2-GW/ EmGFP-miR) provides a rapid and efficient way to clone ds oligo duplexes encoding a desired miRNA target sequence into a vector containing a Pol II promoter (CMV) for use in RNAi analysis. The BLOCK-iT™ Pol II miR RNAi Expression Vector is specifically designed to allow expression of miRNA sequences and contain specific miR flanking sequences that allow proper processing of the miRNA. o Co-cistronic expression of an Emerald GFP (EmGFP) reporter in the pcDNA™6.2-GW/EmGFP-miR vector, resulting in very strong correlation of EmGFP expression with the knockdown activity of your miRNA o The pDONR™221 vector to transfer the premiRNA expression cassette into the lentiviral expression plasmid using Gateway™ Technology. The Gateway™ Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your DNA sequence of interest into multiple vector systems. o A pLenti6/V5-DEST destination vector into which the pre-miRNA cassette from the expression clone is transferred using Gateway™ Technology.This expression plasmid contains elements that allow packaging of the construct into virions and the Blasticidin resistance marker for selection of stably transduced cell lines. o Gateway™ BP and LR Clonase™ II Enzyme Mixes that facilitate the transfer of the pre-miRNA expression cassette from the expression vector into the pLenti6/V5-DEST destination vector. o Components of the ViraPower™ Lentiviral System for production of a replication-incompetent lentivirus that stably expresses the miRNA of interest in both dividing and non-dividing mammalian cells. The ViraPower™ Lentiviral Technology facilitates highly efficient, in vitro or invivo delivery of a target gene or RNA to dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. Based on the lentikat™ systemdeveloped by Cell Genesys (Dull et al., 1998), the ViraPower™ LentiviralTechnology possesses features which enhance its biosafety while allowing highlevel expression in a wider range of cell types than traditional retroviral systems.