The BLOCK-iT™ Pol II miR RNAi Expression Vector Kits combine the advantages of traditional RNAi vectors (stable expression and the ability to use viral delivery) with capabilities for tissue-specific expression and multiple target knockdown from the same transcript. The BLOCK-iT™ Pol II miR RNAi Expression Vector Kits and the BLOCK-iT™ Lentiviral Pol II miR RNAi Expression Systems are designed to express artificial miRNAs which are engineered to have 100% homology to your target sequence and will result in target cleavage. Using Invitrogen’s award-winning BLOCK-iT™ RNAi Designer, over 70% of constructs produce more than 70% knockdown. The Invitrogen Pol II expression family of vectors include:
–Strong expression from the CMV immediate early promoter, with the option to use EF-1α, tissue-specific promoters, or other promoters via MultiSite Gateway™ recombination. –Compatibility with many of Invitrogen’s Gateway™ destination (DEST) vectors for gene expression.
–Ability to control the initiation of the RNAi response with a new BLOCK-iT™ Inducible Pol II miR RNAi Expression Vector Kit w/EmGFP (cat. no. K493900). –A new destination vector in the BLOCK-iT™ HiPerform™ Lentiviral PolII miR RNAi Expression System with EmGFP. The new vector contains an mRNA stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) which generate up to 5-fold higher virus titers and EmGFP expression levels. Additionally, blasticidin resistance is expressed from the mouse PGK-1 promoter to avoid shut-down after multiple passages.
–Co-cistronic expression of Emerald GFP (EmGFP), resulting in correlation of EmGFP expression with knockdown from your miR RNAi.
–Expression of more than one engineered miR RNAi sequence on the same transcript, allowing the knockdown of multiple genes simultaneously and the generation of synthetic phenotypes
The new BLOCK-iT™ Inducible Pol II miR RNAi Expression Vector Kit with EmGFP provides the ability to regulate RNAi experiments. This kit contains the pT-REx-DEST30 Gateway™ vector which after simple cloning and shuttling techniques, produces a miR RNAi expression vector suitable for inducible knockdown. The pT-REx-DEST30 Gateway™ vector contains the CMV promoter with two copies of the tetracycline operator (tetO2) sequence allowing high-level and regulated expression.This permits the study of loss of function in a stably transfected cell line even if the gene of interest is essential. Also, induction of miR RNAi expression can be halted so phenotypic changes can be measured during recovery of gene function.
For a variety of expression options, the miR RNAi cassette, which contains EmGFP (pcDNA™6.2-GW/EmGFP-miR vector only), miR flanking regions, and an miRNA homologous to the target of interest, can be readily moved into a variety of DEST vectors. This occurs through Gateway™ recombination reactions in which the miR RNAi cassette is transferred into a pDONR™ vector (BP reaction) and then into a DEST vector (LR reaction) of choice. The HiPerform™ system has the new pLenti6.4/R4R2/V5-DEST vector for high virus titers and EmGFP expression.
The BLOCK-iT™ HiPerform™ Lentiviral PolII miR RNAi Expression System with EmGFP contains the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP plus all the necessary reagents for Gateway™ cloning into the lentiviral expression system, including: pLenti6.4/R4R2/V5-DEST vector, pLenti6.4/CMV/V5-MSGW/lacZ Control vector, pDONR™221, pENTR5’/EF1αp, pENTR5’/CMVp, 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.
