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

Catalog No. K493900
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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 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.

Specifications

Product Type RNAi Expression Vector Kit
Vector BLOCK-iT RNAi Vectors
Format Kit
Includes 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
No. of Reactions 20
Description BLOCK-iT™ Inducible Pol II miR RNAi Expression Vector Kit with EmGFP, 20rxns, Liquid, CMV/TO Promorter, Transfection Delivery Method, Gateway™ Cloning Method, BLOCK-iT RNAi Vectors Type, Inducible System, GFP (EmGFP) Reporter,
For Use With (Application) RNAi, epigenetics and non-coding RNA research, vector-based RNAi
Gene GFP (EmGFP)
Content And Storage BLOCK-iT™ Inducible PolII miR RNAi Expression Vector Kit w/EmGFP
A cloning box with:
• Linearized pcDNA™6.2-GW/EmGFP-miR
• Annealing buffer (10X)
• T4 DNA Ligase
• DNA Ligation buffer (5X)
• lacZ control oligo
• lacZ control plasmid
• Negative control plasmid
• DNase/RNase-free water
• Forward and reverse sequencing primers

pT-REx-DEST30 Gateway™ Vector Kit
• pT-REx-DEST30
• pT-REx/GW-30/lacZ vectors

T-REx™ Regulatory Module
• pcDNA™6/TR
• Tetracycline

The Gateway™ LR and BP Clonase™ kits, blasticidin, and pDONR™ 221 are also 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.
Cloning Method Gateway™
Constitutive or Inducible System Inducible
Delivery Type Transfection
RNAi Type miRNA
Selection Agent (Eukaryotic) Blasticidin
Promoter CMV/TO
Product Line BLOCK-iT
Quantity 20 Reactions
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Frequently Asked Questions (FAQs)

What are the benefits of the BLOCK-iT miR RNAi expression system over similar systems?

The BLOCK-iT miR RNAi expression system allows you to take advantage of promoter flexibility by choosing from a variety of Pol II promoters like CMV, Ubc, tissue specific, or inducible promoters. The miRNA vectors also allow you to clone multiple sequences in the same vector, thereby enabling you to target more than one gene or more than one location in a gene using a single plasmid. An additional advantage offered by some of the miRNA expression vectors is that transfection efficiency can be monitored with the EmGFP fusion partner.
How can I tell which sequence will be a potent miRNA sequence?

We offer a free online RNAi design program to help you design an effective miRNA sequence. The RNAi designer can be found at https://rnaidesigner.invitrogen.com/rnaiexpress/, or search "RNAi Designer" from the homepage of thermofisher.com.
Can I target multiple genes or multiple locations within a gene using the BLOCK-iT miR RNAi expression system?

Yes. The miRNA cloning vectors are designed such that you can easily chain together multiple miRNAs to express them in one transcript. This can be used to increase transcription levels of the same miRNA sequence, or to combine multiple different sequences in one co-cistronic expression cassette.
Are the BLOCK-iT miR RNAi Expression Kits compatible with adenoviral expression systems?

Yes. The miR miRNA vectors are Gateway cloning compatible, and you could use Gateway cloning to transfer the miR miRNA expression cassette to any of our Gateway-adapted viral expression vectors.
How do the BLOCK-iT miR RNAi Kits work? Do they cleave the specific mRNA target or just suppress translation? Can this system be used for analyzing endogenous miRNAs?

The miRNA vectors use endogenous miRNA processing machinery to allow targeted RNAi knockdown of specific genes, but they are not tested for use in the analysis of endogenous miRNA. Endogenous miRNAs are typically not 100% homologous to their targets and thus invoke translational suppression. Our products are designed with 100% homology to the target gene and generally result in target mRNA cleavage.
How do the BLOCK-iT shRNA products compare to the BLOCK-iT miR RNAi system?

Both systems are used for gene targeting or gene knockdown but each has distinctive features. The shRNA expression vectors like pENTR/U6 or pENTR/H1-TO use Pol III promoters, whereas the miRNA expression vectors are flexible to use more common and more processive Pol II promoters like CMV, EF1 or other mammalian expression promoters. You can only clone a single shRNA sequence into an shRNA vector to target a single gene, whereas multiple miRNA sequences can be cloned together into an miRNA vector to target one or more genes, or multiple locations in a gene. An additional feature of the miRNA expression vectors is that, due to use of Pol II promoters, the miRNA can be expressed directly in fusion with a reporter gene like EmGFP to monitor transfection and transcription.
What are the different promoter choices available with the BLOCK-iT miR RNAi expression system?

All the BLOCK-iT miR RNAi expression vectors are Gateway-adapted and contain the CMV promoter. If more specialized expression is required with a different promoter, the miRNA vectors allow for easy recombination with any other suitable destination vector that Thermo Fisher Scientific offers. A wide variety of Gateway Destination vectors that contain promoters such as EF-1apha or Ubc are available, and all be used for miRNA expression. MultiSite Gateway Technology vectors are also available which will enable you to use a tissue-specific promoter or a promoter of your choice to express miRNA.
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 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.

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