Generation of HIV‐1‐Based Lentiviral Vector Particles

Ali Ramezani1, Robert G. Hawley2

1 American Red Cross, Rockville, Maryland, 2 The 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

  • 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 recipe100× (1 mM) stock (see recipe)]
  • Lentiviral vector plasmid DNA containing the gene of interest (see unit 16.21; Ramezani et al., ; Vigna and Naldini, )
  • Lentiviral packaging construct DNA (e.g., pCMVΔR8.91; Zufferey et al., )
  • VSV‐G expressing plasmid DNA (e.g., pMD.G; Naldini et al., )
  • recipe2.5 M CaCl 2 (see recipe)
  • recipe2× HEPES‐buffered saline (HeBS; see recipe)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 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 protocol 1, step )
  • recipe500 mM sodium butyrate stock solution (see recipe)
  • recipe293T cell growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Additional reagents and equipment for contransfection of 293T cells and preparation of lentiviral supernatants (see protocol 1)

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

  Materials
  • Vector‐containing supernantants (see protocol 1Basic Protocols 1 and protocol 22)
  • 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

  • 100 mg/ml poly‐L‐lysine hydrobromide (PLL; mol. wt. based on viscosity, 27,400) in PBS (see appendix 22 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 protocol 3), resuspended in PBS
  • Phosphate‐buffered saline (PBS; appendix 22) 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 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)
  • recipe293T cell growth medium (see recipe)
  • Vector preparations ( protocol 3Basic Protocols 3 or protocol 54 or protocol 4Alternates Protocol 1 or protocol 62)
  • 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, ), Xgal staining for lacZ (β‐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 ( protocol 3Basic Protocols 3 or protocol 54 or protocol 4Alternates Protocol 1 or protocol 62)
  • recipe293T 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 protocol 7, steps and )

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

  • 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., pCMVΔR8.91)
  • Additional reagents and equipment for flow cytometric analysis of GFP expression (Rasko, )

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

  • p24 ELISA assay kit (e.g., Alliance HIV‐1 p24 ELISA kit from Perkin‐Elmer Life Sciences; cat. no. NEK050001KT)
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Figures

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Literature Cited

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