Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell–Based Systems

Susan Swift1, James Lorens1, Philip Achacoso2, Garry P. Nolan2

1 Rigel, Inc., South San Francisco, California, 2 Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.17C
DOI:  10.1002/0471142735.im1017cs31
Online Posting Date:  May, 2001
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Abstract

This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate‐mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion‐containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be “pseudotyped” with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV‐G‐pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.

     
 
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Table of Contents

  • Basic Protocol 1: Transient Transfection of the Phoenix Retroviral Packaging Line by Calcium Phosphate/DNA Precipitation
  • Alternate Protocol 1: Pseudotyping Retroviral Virions with Vesicular Stomatitis Virus G Protein
  • Support Protocol 1: Concentration and Cryopreservation of VSV‐G‐Pseudotyped Retrovirus
  • Support Protocol 2: Culture of Phoenix Cell Lines
  • Basic Protocol 2: Infection of Adherent Cells
  • Alternate Protocol 2: Spin Infection of Adherent Cells
  • Alternate Protocol 3: Spin Infection of Suspension Cells
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Transient Transfection of the Phoenix Retroviral Packaging Line by Calcium Phosphate/DNA Precipitation

  Materials
  • Exponentially growing packaging cell line in a 75‐cm2 tissue culture flask (either Phoenix‐ecotropic or Phoenix‐amphotropic; see protocol 4)
  • PBS without Ca2+ or Mg2+ (e.g., appendix 2A)
  • 0.05% (w/v) trypsin/0.53 mM EDTA
  • Complete medium: Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) heat‐inactivated FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L‐glutamine
  • 50 mM chloroquine in PBS (stored at −20°C in 1‐ml aliquots)
  • recipe2 M CaCl 2 (see recipe)
  • 1 to 2 mg/ml retroviral vector plasmid DNA (see ), purified by CsCl gradient (unit 10.3) or Qiagen kit
  • recipe2× HeBS, pH 6.99 (see recipe)
  • 6‐cm tissue culture dishes
  • 15‐ml sterile polypropylene conical screw‐cap tubes
  • Additional reagents and equipment for counting cells ( appendix 3C)

Alternate Protocol 1: Pseudotyping Retroviral Virions with Vesicular Stomatitis Virus G Protein

  • Phoenix‐gp cell line (see protocol 4)
  • Vesicular stomatitis virus G (VSV G) expression plasmid: e.g., pME‐VSVG (Lorens and Nolan, unpubl.) or pHCMV G (Yee et al., )
  • Additional reagents and equipment for concentrating and cryopreserving VSV‐G‐pseudotyped retrovirus (see protocol 3)

Support Protocol 1: Concentration and Cryopreservation of VSV‐G‐Pseudotyped Retrovirus

  Materials
  • Retroviral supernatant from VSV‐G‐pseudotyped Phoenix cells (see protocol 2)
  • recipeTSE solution (see recipe)
  • 0.45‐µm filter (e.g., Gelman Sciences syringe filter, acrodisc, Tuffryn membrane)
  • Beckman L series ultracentrifuge (or equivalent)
  • Beckman SW41 or SW28 rotor and polyallomer tubes (rinsed with 70% ethanol and dried in a laminar flow hood)

Support Protocol 2: Culture of Phoenix Cell Lines

  Materials
  • Phoenix‐ecotropic, Phoenix‐amphotropic, and/or Phoenix‐gp cell line (G.P. Nolan; www.stanford.edu/group/nolan/)
  • Complete medium: Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) heat‐inactivated FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L‐glutamine
  • PBS without Ca2+ or Mg2+ (e.g., appendix 2A)
  • 0.05% (w/v) trypsin/0.53 mM EDTA
  • Freezing medium, ice cold: 90% heat‐inactivated FBS/10% (v/v) dimethyl sulfoxide (DMSO)
  • 50 mg/ml hygromycin B in PBS (Boehringer Mannheim)
  • 2 mg/ml diptheria toxin in sterile H 2O (Calbiochem)
  • 75‐cm2 tissue culture flasks
  • Low‐speed centrifuge with rotor and tube adaptors for 50‐ml conical screw‐cap tubes
  • 2‐ml cryogenic vials
  • Insulated box or Cryo 1°C Freezing Container (Nalgene)
  • Additional reagents and equipment for counting cells ( appendix 3C)

Basic Protocol 2: Infection of Adherent Cells

  Materials
  • NIH 3T3 cells
  • Fibroblast growth medium: e.g., Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) heat‐inactivated donor bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L‐glutamine
  • Phoenix‐generated retroviral supernatant: ecotropic or amphotropic (see protocol 1) or pseudotyped (see protocol 2); freshly harvested or frozen and thawed (see protocol 4), or concentrated (see protocol 3)
  • 4 mg/ml polybrene (hexadimethrine bromide; Sigma) in PBS or 5 mg/ml protamine sulfate (Sigma) in PBS (both filtered through a 0.2‐µm filter and stored at 4°C)
  • 6‐cm tissue culture dishes
  • 0.45‐µm filter (optional; Corning bottle‐top cellulose acetate or Gelman Sciences syringe filter, acrodisc, Tuffryn membrane)

Alternate Protocol 2: Spin Infection of Adherent Cells

  • Beckman GS‐6KR or Sorvall RT‐3000B centrifuge with microplate carriers (or equivalent)

Alternate Protocol 3: Spin Infection of Suspension Cells

  • Exponentially growing Jurkat cells (ATCC)
  • Complete RPMI‐1640: RPMI‐1640 medium with 10% heat‐inactivated FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L‐glutamine
  • Beckman GS‐6KR or Sorvall RT‐3000B centrifuge with microplate carriers (or equivalent)
  • 24‐well tissue culture dishes
  • 32°C incubator
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.stanford.edu/group/nolan/
  Nolan laboratory's Web site, which provides protocols, tutorials, programs, and links to other WWW sites. Material transfer forms for Phoenix cells can be downloaded directly. In particular, the development and application of genetic screens is discussed in depth at the site, as are recent updates on the uses of these cells.
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