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Invitrogen™ BLOCK-iT™ Lentiviral Pol II miR RNAi Expression System with EmGFP
Description
Broadens 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 pcDNA6.2-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 in vivo delivery of a target gene or RNA to dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. Based on the lentikat™ system developed by Cell Genesys (Dull et al., 1998), the ViraPower Lentiviral Technology possesses features which enhance its biosafety while allowing high-level expression in a wider range of cell types than traditional retroviral systems.
Cloning, Gateway Cloning, Lentiviral shRNA and miRNA, RNAi, RNAi, Epigenetics and Non-Coding RNA Research, Virus-Based RNAi
Specifications
Specifications
| Product Type | RNAi Expression Vector Kit |
| Gene | GFP (EmGFP) |
| Content And 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. |
| Cloning Method | Gateway™ |
| Constitutive or Inducible System | Constitutive |
| Delivery Type | Lentiviral |
| RNAi Type | miRNA |
| Selection Agent (Eukaryotic) | Blasticidin |
| Vector | pLenti |
| Promoter | CMV |
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For Research Use Only. Not for use in diagnostic procedures.
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