BLOCK-iT™ Pol II miR RNAi Expression Vector Kit

Catalog number: K493500

Invitrogen™  Related applications: Gateway Cloning | RNAi

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Description

The BLOCK-iT™ Pol II miR RNAi Expression Vector Kit combines 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 pcDNA™6.2-GW/miR vector included in the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit is designed to express artificial miRNAs which are engineered to have 100% homology to your target sequence and will result in target cleavage. This vector includes flanking and loop sequences from an endogenous miRNA which directs the excision of the engineered miRNA from a longer Pol II transcript (pre-miRNA). Using Invitrogen's award-winning BLOCK-iT™ RNAi Designer, over 70% of constructs produce more than 70% knockdown. Pol II expression of engineered miRNAs enables:
o Strong expression from the CMV immediate early promoter, with the option to use tissue-specific or other regulated promoters via MultiSite Gateway® recombination
o Compatibility with many of Invitrogen's Gateway® destination (DEST) vectors for gene expression; including Lentiviral vectors for stable transduction of dividing, non-dividing and primary cell types, the Flp-In™ system for single-site chromosomal integration, and alternative reporter fusion constructs
o Expression of more than one engineered miRNA on the same transcript, allowing the knockdown of multiple genes simultaneously and the generation of synthetic phenotypes
How it Works
For high levels of expression of your miR RNAi sequence, the pcDNA™6.2-GW/miR vector includes the CMV promoter (Figure 1). Simply input a RefSeq accession number or a nucleotide sequence into the free online BLOCK-iT™ RNAi Designer and the software will design optimized miRNAs that have 100% homology to the target of interest. Clone the miRNA into the vector using a fast ligation protocol and transfect for immediate miRNA expression. Expressed miRNA is processed by the endogenous cellular machinery in the nucleus (including Drosha) and then transported into the cytoplasm where it is further processed by Dicer (Figure 2). The fully processed miRNA is then incorporated into RISC where it functions like an siRNA and results in cleavage of the mRNA target.
For a variety of expression options, the miRNA cassette, 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 miRNA cassette is transferred into a pDONR™ vector (BP reaction) and then into a DEST vector (LR reaction) of choice .
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Promoter: CMV
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: Constitutive

Contents & storage

The BLOCK-iT™Pol II miR RNAi Expression Vector Kit contains two boxes. The cloning box contains linearized pcDNA™6.2-GW/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.

Documents

Manuals & protocols