ViraPower™ HiPerform™ Promoterless Gateway® Expression System

Catalog number: A11145

Invitrogen™  Related applications: Protein Expression

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Description

The ViraPower™ HiPerform™ Promoterless Gateway® Expression System includes all the components needed to generate lentivirus, including vector kit, 293FT cell line, and the support kit. This kit combines Invitrogen’s 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 promoterless 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
• ViraPower™ HiPerform™ Promoterless Gateway® Vector Kit (Cat # A11146)
• ViraPower™ Bsd Lentiviral Support Kit (Cat # K497000)
• 293FT Cell Line (Cat # R70007)
• Gateway® LR Clonase® II Plus Enzyme Mix (Cat # 12538120)

For research use only. Not intended for any therapeutic or diagnostic use.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Promoter: CMV, EF-1α, None (Promoterless)
Key Function: Clone your own Promoter, Viral Expression
Protein Tag or Fusion: V5 Epitope Tag
Delivery Method: Lentiviral
Cloning Method: Gateway®
Selection Agent (Eukaryotic): Blasticidin
Vector Type: pLenti
Antibiotic Resistance (Bacterial): Ampicillin (AmpR), Blasticidin (BsdR)
Cleavage: No Cleavage Site
Comments: Use the provided EF1&alpha or CMV promoter, or clone your own promoter of interest.
Constitutive or Inducible System: Constitutive
Gateway Enzyme: LR Clonase® II, LR Clonase® II Plus
PCR Enzyme: Proof-Reading Polymerase
Product Size: 20 reactions
Protein Tag Position (to your gene): C-terminal
Replication Origin: pUC Origin
Selection Marker (Eukaryotic): BsdR
Selection Marker Promoter: PGK Promoter
Special Elements: WPRE, cPPT

Contents & storage

Contents
The ViraPower™ HiPerform™ Promoterless Gateway® Expression System includes the following components:

ViraPower™ HiPerform™ Promoterless Gateway® Vector Kit (Cat # A11146) which includes:
• pLenti6.4⁄R4R2⁄V5-DEST™ Kit which includes:
  • 6 ug of pLenti6.4⁄R4R2⁄V5-DEST™ vector (40 µL of vector at 150 ng⁄µL in TE Buffer, pH 8.0)
  • 10 ug of pLenti6.4⁄CMV⁄V5-MSGW⁄lacZ plasmid (20 µL of vector at 0.5 µg⁄µL in TE Buffer, pH 8.0)
  • 10 ug of pENTR™ 5’⁄CMVp plasmid (20 µL of plasmid at 0.5 µg⁄µL in TE Buffer, pH 8.0)
  • 10 ug of pENTR™ 5’⁄EF1αp plasmid (20 µL of plasmid at 0.5 µg⁄µL in TE Buffer, pH 8.0)

  • pENTR™ 5’ TOPO® TA Cloning Kit (Cat # K59120) which includes:
  • pENTR™ 5´-TOPO® TA Cloning® Reagents, including pENTR™ 5´-TOPO® Vector
  • One Shot® TOP10 Chemically Competent E. coli (C4040-03)
  • One Shot® Stbl3™ Chemically Competent E. coli (Cat # C737303): 21 X 50 µL of Stbl3™ Cells, 6 mL of S.O.C. Medium and 50 µL of pUC19 control DNA (10 pg⁄µL)
  • pENTR™ Gus positive control plasmid

  • ViraPower™ Bsd Lentiviral Support Kit (Cat # K497000) which includes:
  • 195 ug of ViraPower™ Packaging Mix (Contains 195 µL of a mixture of pLP1, pLP2, and pLP⁄VSVG plasmids, at 1 ug⁄µL in TE Buffer, pH 8.0)
  • 0.75 ml of Lipofectamine-® 2000 (Cat # 11668027)
  • 50 mg of Blasticidin powder (Cat # R21001)
  • 293FT Cell Line (Cat # R70007): 1 vial containing 3 X 106 frozen cells in 1 mL of Freezing Medium
  • Gateway® LR Clonase® II Plus Enzyme Mix (Cat # 12538120): 40 µL of Gateway® LR Clonase® II Plus Enzyme Mix and 40 µL of Proteinase K Solution at a concentration of 2 ug⁄mL

  • Storage
    • pLenti6.4⁄R4R2⁄V5-DEST™ Kit: -20°C
    • pENTR™ 5’ TOPO® Reagents: -20°C
    • ViraPower™ Packaging Mix: -20°C
    • LR Clonase® II Plus Enzyme Mix: -80°C
    • Blasticidin: -20°C
    • Lipofectamine™ 2000: 4°C (do not freeze)
    • One Shot® Stbl3™ Chemically Competent E.coli: -80°C
    • One Shot® TOP10 Chemically competent E.coli: -80°C
    • 293FT Cell Line: Liquid Nitrogen
    • pENTR™ Gus positive Control: -20°C

    Documents

    Manuals & protocols