Invitrogen™ ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit

Catalog No.	Quantity
A11146	20 Reactions

Catalog No. A11146 Supplier Invitrogen™ Supplier No. A11146

Description

The ViraPower HiPerform Promoterless Gateway Vector Kit contains MultiSite Gateway-adapted ViraPower HiPerform promoterless lentiviral expression vector, pLenti6.4/R4R2/V5-DEST for easy recombination-based cloning and expression of a target gene in dividing and non-dividing mammalian cells.

The ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit contains the MultiSite Gateway™-adapted ViraPower™ HiPerform™ promoterless lentiviral expression vector, pLenti6.4/R4R2/V5-DEST™ for easy recombination-based cloning and lentiviral-based high-level expression of a target gene from any promoter of choice in dividing and non-dividing mammalian cells. The pLenti6.4/R4R2/V5-DEST™ vector is equipped with two key genetic elements, making it a HiPerform™ vector: the Woodchuck Posttranscriptional Regulatory Element (WPRE) and the central Polypurine Tract (cPPT) sequence from the HIV-1 integrase gene to produce at least 4-fold increase in protein expression compared to vectors lacking these elements.

Advantages

• Generates replication-incompetent lentivirus for transducing dividing and non-dividing mammalian cells
• Easy, simultaneous, recombination-based cloning of multiple DNA fragments in a defined order and orientation using MultiSite Gateway™ technology
• Expression of the target gene under the control of a promoter of choice
• Stable, long-term expression
• Enhanced protein expression, up to 4-fold or greater, compared to traditional lentiviral expression systems

Key Features

• WPRE from the woodchuck hepatitis virus, increases transgene expression and cPPT from the HIV-1 integrase gene, increases the copy number of lentivirus integrating into the host genome, thus increasing viral titer. WPRE and cPPT together produce at least a four-fold increase in protein expression in most cell types, compared to other vectors that do not contain these elements.
• Promoterless vector to express the target gene under the control of a promoter of choice
• Blasticidin selection marker for stable selection under control of PGK promoter for long-term, persistent expression

Kit includes

• pLenti6.4/R4R2/V5-DEST™ Kit
• pENTR™ 5' TOPO™ TA Cloning Kit (Cat. No. K59120)
• One Shot™ Stbl3™ Chemically Competent E. coli (Cat. No. C737303)
• pENTR™ Gus positive control plasmid

Specifications

• Constitutive or Inducible System: Constitutive
• Delivery Type: Lentiviral
• Promoter: None (Promoterless), EF-1α, CMV
• Product Type: Vector Kit
• Selection Agent (Eukaryotic): Blasticidin
• Content And Storage:

  pLenti6.4/R4R2/V5-DEST™ Kit
  • pLenti6.4/R4R2/V5-DEST™ vector (40 μL of vector at 150 ng/μL in TE Buffer, pH 8.0), 6 μg
  • pLenti6.4/CMV/V5-MSGW/lacZ plasmid (20 μL of vector at 0.5 μg/μL in TE Buffer, pH 8.0), 10 μg
  • pENTR 5'/CMVp plasmid (20 μL of plasmid at 0.5 μg/μL in TE Buffer, pH 8.0), 10 μg
  • pENTR 5'/EF1αp plasmid (20 μL of plasmid at 0.5 μg/μL in TE Buffer, pH 8.0), 10 μg
  Store at -20°C
  
  pENTR 5' TOPO™ TA Cloning Kit (Cat. No. K95120)
  • pENTR 5' TOPO™ Reagents, store at -20°C.
  • One Shot™ TOP10 Chemically Competent E.coli (Cat. No. C404003), store at -80°C.
  
  One Shot™ Stbl3™ Chemically Competent E. coli (Cat. No. C737303)
  • Stbl3™ Cells, 6 mL of S.O.C. Medium, 21 x 50 μL
  • pUC19 control DNA (10 pg/μL), 50 μL
  Store at -80°C
  
  pENTR™ Gus positive control plasmid, store at -20°C

• Antibiotic Resistance Bacterial: Ampicillin (AmpR), Blasticidin (BsdR)
• Cleavage: No Cleavage Site
• Gateway Enzyme: LR Clonase™ II Plus, LR Clonase™ II
• PCR Enzyme: Proof-Reading Polymerase
• Protein Tag Position (to your gene): C-terminal
• Replication Origin: pUC Origin
• Selection Marker Promoter: PGK Promoter
• Selection Marker Eukaryotic: BsdR
• Special Elements: WPRE, cPPT
• Protein Tag: V5 Epitope Tag
• Cloning Method: Gateway
• Quantity: 20 Reactions
• Vector: pLenti
• Product Line: Gateway, HiPerform, ViraPower
• For Use With (Application): Clone your own Promoter, Viral Expression

Frequently Asked Questions (FAQs)

Do 293FT cells lift off the plate during lentivirus production?

If 293FT cells detach shortly after transfection (4 hours to overnight):

- This may be a sign of Lipofectamine 2000 toxicity. Cells may have been plated too sparsely prior to transfection.
- The cells may not have been handled gently enough (these cells have a tendency to lift off easily).
- The cells may have been kept at room temperature for too long.

If cells detach 48 to 72 hours post-transfection:
- If the cells lift off in large sheets, this may be a sign of lentivirus production.

What are syncytia?

Syncytia are large multi-nucleated cells that result from VSV-G-induced fusion with neighboring 293FT producer cells. Syncytia production is indicative of high transfection efficiency and lentivirus production. Keep in mind, though, that the absence of syncytia does not mean that virus will not be produced.

Why do I have to culture 293FT cells under Geneticin antibiotic selection?

The 293FT cell line stably expresses the SV40 large T antigen from the pCMVSPORT6Tag.neo plasmid that contains the neomycin resistance marker. In order to maintain the plasmid/phenotype, the cells have to be routinely cultured in medium containing Geneticin (G418) antibiotic at a concentration of 500 µg/mL.

Do you recommend a specific FBS for culturing 293FT cells? Which plastic plates do you recommend?

We use Mycoplasma-tested Gibco FBS (Cat. No. 16000-044). We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources. We use the following plasticware for 293FT cells:

T175-Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 µm vented plug seal cap.
T75-Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 µm vented seal cap.
100 mm plate-Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate.
We get excellent adherence on these plates under routine cell culture/maintenance conditions.

Do you offer a promoterless lentiviral vector where I can introduce my own promoter?

We offer the ViraPower HiPerform Promoterless Gateway Expression System (Cat. No. A11145) that combines ViraPower HiPerform Lentiviral and MultiSite Gateway technologies to facilitate easy recombination-based cloning and lentiviral-based high-level expression of a target gene from any promoter of choice in dividing and non-dividing mammalian cells. The ViraPower HiPerform Promoterless GatewayVector Kit is available separately (Cat. No. A11146).

Which competent E. coli do you recommend using for propagation of my Gateway-adapted mammalian Destination vector?

We recommend using One Shot ccdB Survival 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha, for propagation and maintenance, as these strains are sensitive to ccdB effects.

Do I need to include a consensus Kozak sequence when I clone my gene of interest into one of your mammalian expression vectors?

The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

What is the consensus Kozak sequence and what is the function of the Kozak sequence?

Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

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