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Generation of HIV‐1‐Based Lentiviral Vector Particles

Ali Ramezani1,  Robert G. Hawley1,2

1American Red Cross, Rockville, Maryland
2The George washington University, Washington, D.C.


Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 16.22
DOI: 
10.1002/0471142727.mb1622s60
Online Posting Date: 
November, 2002
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Abstract

This unit presents protocols outlining the methodology and techniques involved in the construction and application of HIV-1-based lentiviral vector systems. Also described are procedures that can be used to concentrate and purify high-titer recombinant lentiviral vector preparations, as well as protocols for transduction of adherent and suspension cells.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Cotransfection of 293T Cells and Production of Lentiviral Vector Supernatants
  • Basic Protocol 2: Stimulation of Vector Particle Production Using Sodium Butyrate
  • Basic Protocol 3: Concentration of HIV-1 Vector Particles by Ultracentrifugation
  • Alternate Protocol 1: Concentration of HIV-1 Vector Particles by Poly-L-Lysine Precipitation
  • Basic Protocol 4: Purification of Concentrated Lentiviral Vector Particles by Anion-Exchange Chromatography
  • Alternate Protocol 2: Purification of Concentrated Lentiviral Vector Particles by the Dual-Filter Method
  • Basic Protocol 5: Titration of Lentiviral Vector Stocks
  • Basic Protocol 6: Detection of Replication-Competent Viruses by the Reverse Transcriptase Assay
  • Alternate Protocol 3: Detection of Replication-Competent Viruses by the Tat-Induction Assay
  • Alternate Protocol 4: Detection of Replication-Competent Viruses by the Gag Transfer Assay
  • Basic Protocol 7: Transduction of Target Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Cotransfection of 293T Cells and Production of Lentiviral Vector Supernatants

 Materials
  • 293T human embryonic kidney cell line (ATCC #CRL-11268)
  • 293T cell growth medium (see recipe) with and without 10 mM HEPES [add HEPES from 100× (1 mM) stock (see recipe)]
  • Lentiviral vector plasmid DNA containing the gene of interest (see UNIT 16.21; Ramezani et al., 2000; Vigna and Naldini, 2000)
  • Lentiviral packaging construct DNA (e.g., pCMVR8.91; Zufferey et al., 1997)
  • VSV-G expressing plasmid DNA (e.g., pMD.G; Naldini et al., 1996)
  • 2.5 M CaCl2 (see recipe)
  • 2× HEPES-buffered saline (HeBS; see recipe)
  • Phosphate-buffered saline (PBS; APPENDIX 2)
  • 100-mm tissue culture dishes
  • Tabletop centrifuge
  • 0.45-µm pore-size filter

Additional reagents and equipment for cell culture and counting cells (APPENDIX 3F)

Basic Protocol 2: Stimulation of Vector Particle Production Using Sodium Butyrate

 Materials
  • Cells containing calcium phosphate–DNA coprecipitate (see Basic Protocol 1, step )
  • 500 mM sodium butyrate stock solution (see recipe)
  • 293T cell growth medium (see recipe)
  • Phosphate-buffered saline (PBS; APPENDIX 2)
  • Additional reagents and equipment for contransfection of 293T cells and preparation of lentiviral supernatants (see Basic Protocol 1)

Basic Protocol 3: Concentration of HIV-1 Vector Particles by Ultracentrifugation

 Materials
  • Vector-containing supernantants (see Basic Protocols 1 and 2)
  • Appropriate medium to resuspend concentrated vector particles
  • Ultracentrifuge and rotor
  • Polycarbonate 70-ml ultracentrifuge bottles and caps
  • Tabletop centrifuge

Alternate Protocol 1: Concentration of HIV-1 Vector Particles by Poly-L-Lysine Precipitation

 Additional Materials (also see Basic Protocol 3)
  • 100 mg/ml poly-L-lysine hydrobromide (PLL; mol. wt. based on viscosity, 27,400) in PBS (see APPENDIX 2 for PBS), freshly prepared
  • Appropriate medium to resuspend concentrated vector particles
  • 250-ml centrifuge bottles

Basic Protocol 4: Purification of Concentrated Lentiviral Vector Particles by Anion-Exchange Chromatography

 Materials
  • Vector particles concentrated by ultracentrifugation (see Basic Protocol 3), resuspended in PBS
  • Phosphate-buffered saline (PBS; APPENDIX 2) containing 1 M NaCl
  • 80 × 6–mm anion-exchange column: Fractoflow 80-6 column (Merck) packed with 40- to 90-µm mesh size resin according to manufacturer's instructions (also see UNIT 10.10)
  • Ultrafree-15 centrifugal filter devices (Millipore; 100,000 MWCO; also see APPENDIX 3C)

Alternate Protocol 2: Purification of Concentrated Lentiviral Vector Particles by the Dual-Filter Method

 Materials
  • Vector supernantants concentrated by ultracentrifugation (see Basic Protocol 3)
  • Appropriate serum-free medium
  • 10-ml syringes and 0.22-µm syringe filters
  • Ultrafree-15 centrifugal filter devices (Millipore; 100,000 MWCO; also see APPENDIX 3C)

Basic Protocol 5: Titration of Lentiviral Vector Stocks

 Materials
  • HT1080 cell line (ATCC #CCL-121)
  • 293T cell growth medium (see recipe)
  • Vector preparations (Basic Protocols 3 or 4 or Alternates Protocol 1 or 2)
  • 6 mg/ml (1000×) polybrene stock solution
  • G418 (UNIT 9.5)
  • 0.3% (w/v) crystal violet in 70% (v/v) methanol
  • 6-well tissue culture plates
  • Additional reagents and equipment for flow cytometric analysis of EGFP expression (Rasko, 1999), Xgal staining for lacZ (-galactosidase) expression (UNIT 9.10), or enumeration of neo-resistant cells (UNIT 9.5)

Basic Protocol 6: Detection of Replication-Competent Viruses by the Reverse Transcriptase Assay

 Materials
  • HT1080 cell line (ATCC #CCL-121)
  • Undiluted vector particle supernatant (Basic Protocols 3 or 4 or Alternates Protocol 1 or 2)
  • 293T cell growth medium (see recipe)
  • 35-, 100-, and 150-mm tissue culture dishes
  • Tabletop centrifuge
  • Ultracentrifuge and rotor
  • Reverse transcriptase assay kit (e.g., Roche Diagnostics, cat. no. 1468120)

Additional reagents and equipment for stable transduction of cells (see Basic Protocol 5, steps and )

Alternate Protocol 3: Detection of Replication-Competent Viruses by the Tat-Induction Assay

 Additional Materials (also see Basic Protocol 6)
  • CEM-GFP cell line (NIH AIDS Research and Reference Reagent Program)
  • Growth medium for CEM-GFP cells: IMDM containing 10% FBS (see recipe for serum-containing growth media for transduction)
  • 6 mg/ml polybrene stock solution
  • HIV packaging plasmid DNA (e.g., pCMVR8.91)
  • Additional reagents and equipment for flow cytometric analysis of GFP expression (Rasko, 1999)

Alternate Protocol 4: Detection of Replication-Competent Viruses by the Gag Transfer Assay

 Additional Materials (also see Basic Protocol 6)
  • p24 ELISA assay kit (e.g., Alliance HIV-1 p24 ELISA kit from Perkin-Elmer Life Sciences; cat. no. NEK050001KT)
Looking for materials?  Suppliers Guide
     
 
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Literature Cited

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