Invitrogen™

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

Catalog number:  K493800

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 Type: Lentiviral
Product Line: BLOCK-iT™, Gateway®
Promoter: CMV
RNAi Type: miRNA
Reporter Gene: GFP (EmGFP)
Selection Agent (Eukaryotic): Blasticidin
Vector: pLenti

Contents & storage

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

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