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

Catalog No. K493400
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Description

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

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.

Specifications

Product Line BLOCK-iT, HiPerform
Quantity 20 reactions
No. of Reactions 20 Reactions
Product Type Pol II miR RNAi Expression Vector Kit
RNAi Type Expressed RNAi (miRNA, shRNA)
Cloning Method Gateway
Promoter EF-1α, CMV
Protein Tag V5 Epitope Tag
Reporter Gene GFP (EmGFP)
Selection Marker Promoter PGK Promoter
Content And Storage 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.
Selection Agent (Eukaryotic) Blasticidin
Vector pLenti
Constitutive or Inducible System Constitutive
Delivery Type Lentiviral
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Frequently Asked Questions (FAQs)

I'm getting no fluorescence signal with my expression clone containing EmGFP. What should I do?

Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1-3 days before assaying for fluorescence.
I'm seeing nonspecific, off-target gene knockdown. What should I do?

The target sequence used may contain strong homology to other genes; please select a different target region.
I am not getting any colonies after titering. What would suggest I try?

Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene reagent.
I'm getting few or no colonies, even with the transformation control. What could be the cause of this?

Ensure that the competent cells used were stored properly at -80 degrees C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation.

I'm getting differently sized colonies after TOP10 E. coli transformation when using the miRNA lentiviral expression system. Which one should I use?

Some transformants may contain plasmids in which unwanted recombination has occurred between the 5' and 3' LTR. We recommend using our One Shot Stbl3 Chemically Competent E. coli cells, as they help in stabilizing lentiviral DNA containing direct repeats, and generally give rise to fewer unwanted recombinants. We recommend screening both colony sizes; however, in general, for lentiviral plasmids the small colonies tend to be the correct clones. Large colonies may have undergone a recombination event to delete part of the plasmid, thus allowing the cells to grow faster.
I'm seeing a low level of gene knockdown or no gene knockdown. What can you suggest I try?

Low expression levels can be due to several factors. Please see the suggestions below:

- Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
- Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
- Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
- Target region is not optimal: select a different target region.
- Ensure siRNA is designed according to guidelines listed in the respective manual.

I'm seeing cytotoxic effects after transfection of my shRNA/miRNA construct. What is causing this?

You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.
I'm trying to create my entry clone but am seeing mutated inserts. What should I do?

We highly recommend sequencing positive transformants to confirm the sequence of the ds oligo insert. When screening transformants, we find that up to 20% of the clones may contain mutated inserts (generally 1 or 2 bp deletions within the ds oligo). The reason for this is not known, but may be due to triggering of repair mechanisms within E. coli as a result of the inverted repeat sequence within the ds oligo insert. Note: Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.
Mutated inserts could also be caused by using poor-quality single-stranded oligos. Use mass spectrometry to check for peaks of the wrong mass, or order HPLC- or PAGE-purified oligos to avoid this problem.

I'm trying to anneal my oligos to create a ds oligo for ligation into one of your shRNA or miRNA RNAi vectors. When I run my ligated ds oligo on an agarose gel, I do not see any bands representing the ds oligo. What could be happening?

- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5' end, while the bottom-strand oligo should include AAAA on the 5' end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5' end and that the bottom-strand oligo includes CCTG at the 5' end.

I'm trying to anneal my oligos to create a ds oligo for ligation into one of your shRNA or miRNA RNAi vectors. When I run my ligated ds oligo on an agarose gel, the bands are weak. What could be happening?

Please review the possibilities below:

- Single-stranded oligos designed incorrectly; verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top strand oligo.
- Ensure that oligos are annealed at room temp for 5-10 minutes after heating to 95 degrees C.
- Check the molar ratio you are using for annealing top and bottom-strand oligo; equal amounts should be used.

What does cytopathic effect mean?

Adenovirus is not an actively lytic virus, meaning that mature viral particles accumulate in the cell over the course of two to three days. As virus accumulates, the producer cell rounds up and eventually bursts due to the sheer number of virus particles inside. Once this occurs, neighboring cells become infected and the three-day cycle begins again. The term “cytopathic effect”, or CPE, is used to describe this and is typically visible within approximately 7 days posttransfection in the form of “comet-shaped” plaques resulting from two rounds of infection, replication and cell burst.

After 7 days, CPE will expand and eventually take over the plate by approximately 10 days posttransfection.
10 days are required to produce virus from a transfected dish of cells (as just described).
Once an initial viral stock is produced, it can be amplified directly by infection of fresh 293A cells at a multiplicity of infection (MOI) of 3.

What is the difference between infection and transduction?

Please see the definitions below:

Infection: Applies to situations where viral replication occurs and infectious viral progeny are generated. Only cell lines that stably express E1 can be infected.
Transduction: Applies to situations where no viral replication occurs and no infectious viral progeny are generated. Mammalian cell lines that do not express E1 are transduced. In this case, you are using adenovirus as a vehicle to deliver shRNA.

What are the general steps in creating an RNAi viral system?

Please see the steps below:

Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT entry (shRNA) or expression (miR RNAi) vectors.
Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway recombination.
Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at -80 degrees C.
Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired).
Transduce lentiviral or adenoviral stocks to any cell type.

For Research Use Only. Not for use in diagnostic procedures.