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

Catalog number: K493800

Invitrogen™  Related applications: Gateway Cloning | RNAi

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Description

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.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Cloning Method: Gateway®
Constitutive or Inducible System: Constitutive
Delivery Method: Lentiviral
Product Size: 20 reactions
Promoter: CMV
RNAi Type: miRNA
Reporter: GFP (EmGFP)
Selection Agent (Eukaryotic): Blasticidin
Vector Type: pLenti

Contents & storage

The BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP contains either the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit which contains two boxes. The cloning box contains linearized pcDNA™6.2-GW⁄EmGFP-miR, 10X annealing buffer, T4 DNA Ligase, 5X DNA Ligation buffer, lacZ control oligo, lacZ control plasmid, negative control plasmid, DNase/RNase-free water, and forward and reverse sequencing primers. Store the vectors, buffers, control oligo and plasmids, water, and sequencing primers at -20°C. The One Shot® box contains transformation reagents including twenty-one 50-µl aliquots of One Shot® TOP10 Chemically Competent E. coli, S.O.C. medium, and a pUC19 supercoiled control plasmid. Store these transformation reagents -80°C. All reagents are guaranteed stable for 6 months when properly stored.

The BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP also includes all the necessary reagents for Gateway® cloning into the lentiviral expression system, including: pLenti6⁄V5-DEST Vector, pLenti6-V5⁄GW-lacZ control, pDONR™221, ViraPower™ Lentiviral Bsd Support Kit (ViraPower™ Packaging Mix, Lipofectamine™ 2000 transfection reagent, and Blasticidin), 293FT Cell Line, One Shot® Stbl3™ Chemically Competent E. coli, One Shot® TOP10 Chemically Competent E. coli, and Gateway® LR and BP Clonase® II Enzyme Mixes. Store the Lipofectamine™ 2000 reagent at +4°C (do not freeze). Store the vectors, ViraPower™ Packaging Mix, Blasticidin, and both Clonase® II Enzyme Mixes at -20°C. Store the competent cells at -80°C. Store the 293FT Cell Line in liquid nitrogen. All reagents are guaranteed stable for 6 months when properly stored.

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