ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit

Invitrogen™

ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit

Catalog Number	Quantity
A11146	20 Reactions

Catalog number A11146

Price (BRL)

Quantity:

20 Reactions

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

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

Specifications

Antibiotic Resistance BacterialAmpicillin (AmpR), Blasticidin (BsdR)

CleavageNo Cleavage Site

Constitutive or Inducible SystemConstitutive

Delivery TypeLentiviral

For Use With (Application)Clone your own Promoter, Viral Expression

Gateway EnzymeLR Clonase™ II Plus, LR Clonase™ II

PCR EnzymeProof-Reading Polymerase

Product TypeVector Kit

Protein Tag Position (to your gene)C-terminal

Quantity20 Reactions

Replication OriginpUC Origin

Selection Agent (Eukaryotic)Blasticidin

Selection Marker EukaryoticBsdR

Selection Marker PromoterPGK Promoter

Special ElementsWPRE, cPPT

VectorpLenti

Cloning MethodGateway

Product LineGateway, HiPerform, ViraPower

PromoterNone (Promoterless), EF-1α, CMV

Protein TagV5 Epitope Tag

Unit SizeEach

Contents & 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

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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).

View all ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit FAQs