BLOCK-iT™ Inducible Pol II miR RNAi Expression Vector Kit with EmGFP

Catalog number: K493900

Invitrogen™  Related applications: RNAi

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Description

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 tissue-specific or other regulated 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.
–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/RF R2/V5-DEST destination vector for high virus titers and EmGFP expression.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Promoter: CMV⁄TO
Reporter: GFP (EmGFP)
RNAi Type: miRNA
Vector Type: BLOCK-iT RNAi Vectors
Product Size: 20 reactions
Cloning Method: Gateway®
Delivery Method: Transfection
Selection Agent (Eukaryotic): Blasticidin
Constitutive or Inducible System: Inducible

Contents & storage

The BLOCK-iTTM Inducible PolII miR RNAi Expression Vector Kit w/EmGFP contains a cloning box with 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. This kit also contains the pT-REx-DEST30 Gateway® Vector kit (includes pT-REx-DEST30 and pT-REx/GW-30/lacZ vectors), and the T-RExTM Regulatory Module (includes pcDNA™6/TR and Tetracycline). The Gateway® LR and BP Clonase® kits, blasticidin, and pDONRTM221 are included. Store the vectors, buffers, control oligo and plasmids, antibiotics, clonase kits, 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.

Documents

Manuals & protocols