Transient Transfection Methods for Preparation of High‐Titer Retroviral Supernatants

Warren Pear1

1 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.11
DOI:  10.1002/0471142727.mb0911s36
Online Posting Date:  May, 2001
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Abstract

Generation of high‐titer retrovirus by transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high‐titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient transfection has also increased the versatility of retrovirus‐mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus‐transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293‐based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential transfection or cotransfection. Virus produced by transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.

     
 
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Table of Contents

  • Basic Protocol 1: Transient Transfection of a Retrovirus Vector into 293 Cells
  • Support Protocol 1: Growth and Storage of 293 Cells
  • Basic Protocol 2: Pseudotyping a Stable Cell Line Sequentially with VSV G Protein
  • Alternate Protocol 1: Pseudotyping Cotransfectionally with VSV G Protein
  • Basic Protocol 3: Infection of Adherent Cells with Retroviral Supernatant
  • Alternate Protocol 2: Infection of Adherent Cells by Spin Infection
  • Basic Protocol 4: Infection of Nonadherent Cells by Retroviral Supernatant
  • Alternate Protocol 3: Infection of Nonadherent Cells by Cocultivation
  • Alternate Protocol 4: Infection of Nonadherent Cells by Spin Infection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Transient Transfection of a Retrovirus Vector into 293 Cells

  Materials
  • 293 cells or appropriate packaging cell line (see Table 97.80.4711)
  • recipe293 cell growth medium (see recipe)
  • 25 mM chloroquine in PBS (1000×; store at −20°C; optional)
  • Retroviral plasmid DNA
  • 2 M CaCl 2
  • recipe2× HEPES‐buffered saline (HeBS), pH 7.05 (see recipe)
  • 500 mM sodium butyrate, pH 7.0 (50× optional)
  • 60‐mm tissue culture dishes
  • 0.45‐µm filter Sorvall RT‐3000B centrifuge and rotor (or equivalent)
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F) and calcium phosphate transfection (unit 9.1)
NOTE: All solutions should be filtered through a 0.2‐µm filter or autoclaved.

Support Protocol 1: Growth and Storage of 293 Cells

  Materials
  • 293 cells or packaging cell lines (e.g., Bosc23; see Table 97.80.4711) derived from 293 cells
  • recipe293 cell growth medium (see recipe)
  • Freezing medium: 90% (v/v) heat‐inactivated FBS/10% (v/v) DMSO
  • Trypsin solution: 0.05% (v/v) trypsin/0.53 mM EDTA
  • PBS without Ca2+ or Mg2+ ( appendix 22)
  • 10‐cm tissue culture dishes
  • Sorvall RT‐3000B centrifuge and rotor (or equivalent)
  • 2‐ml cryogenic vials

Basic Protocol 2: Pseudotyping a Stable Cell Line Sequentially with VSV G Protein

  Materials
  • Retroviral packaging cell line with amphoteric host range (e.g., PA317, ΨCRIP, GP+Am12, or Bing; see Table 97.80.4711) and appropriate culture medium
  • Retroviral vector DNA
  • gag‐pol‐expressing cell line (e.g., Anjou65, ATCC CRL 11268; see Table 97.80.4711) and appropriate culture medium
  • recipe293 cell growth medium (see recipe)
  • VSV G protein expression plasmid: e.g., pHCMV‐VSV‐G (Matsubara et al., )
  • recipeTNE buffer (see recipe) or 1% (v/v) Hanks basic salt solution (HBSS; appendix 22)
  • 60‐mm tissue culture dishes
  • Sorvall RT‐3000B centrifuge and rotor (or equivalent)
  • Beckman L3‐50 centrifuge and SW 41 rotor (or equivalent)
  • Additional reagents and equipment for culturing of mammalian cells ( appendix 3F), transfecting cells and harvesting virus (see protocol 1), infecting cells (see protocol 5Basic Protocols 3 or protocol 74 or protocol 6Alternate Protocols 2, protocol 83, or protocol 94), selecting clones with drugs (unit 9.5), assaying clones for the gene of interest (e.g., units 9.6 9.8 & 9.10), and freezing cells (see 9.11)

Alternate Protocol 1: Pseudotyping Cotransfectionally with VSV G Protein

  Materials
  • 293 cells or 293‐derived cells expressing retroviral gag‐pol: e.g., Anjou65 (ATCC CRL 11269)
  • 293 cell growth medium (see recipe)
  • Retrovirus vector DNA
  • VSV G protein expression plasmid: e.g., pHCMV‐VSV‐G (Matsubara et al., )
  • 60‐mm tissue culture dishes
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F), modified calcium phosphate–mediated transient transfection of cells (see protocol 1), and concentrating pseudotyped viral supernatants (see protocol 3)

Basic Protocol 3: Infection of Adherent Cells with Retroviral Supernatant

  Materials
  • Retroviral supernatant, fresh or frozen (see protocol 1Basic Protocols 1 or protocol 32)
  • Target cells: e.g., NIH 3T3 cells
  • 4 mg/ml polybrene in PBS ( appendix 22), filtered and stored at 4° or −20°C
  • Fibroblast growth medium
  • Antibiotic for drug selection (unit 9.5; optional)
  • 10‐cm tissue culture dishes
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F) and for assaying of reporter gene expression (e.g., units 9.6 9.8 & 9.10) or drug selection (unit 9.5).

Alternate Protocol 2: Infection of Adherent Cells by Spin Infection

  Materials
  • Target cells: e.g., NIH 3T3 cells
  • Fibroblast growth medium
  • Retroviral supernatant
  • 4 mg/ml polybrene in PBS ( appendix 22), filtered and stored at 4° or −20°C
  • 6‐well tissue culture plates
  • Beckman GS‐6KR or Sorvall RT‐3000B centrifuge with microplate carriers (or equivalent)
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F) and for assaying of reporter gene expression (e.g., units 9.6 9.8 & 9.10) or drug selection (unit 9.5)

Basic Protocol 4: Infection of Nonadherent Cells by Retroviral Supernatant

  Materials
  • Retroviral supernatant
  • 4 mg/ml polybrene in PBS ( appendix 22), filtered and stored at 4° or −20°C
  • Target cell growth medium
  • Exponentially growing nonadherent target cells
  • 15‐ml centrifuge tube
  • 60‐mm tissue culture dishes
  • Sorvall RT‐3000B centrifuge and rotor (or equivalent)
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F) and for assaying of reporter gene expression (units 9.6 9.8 & 9.10) or drug selection (unit 9.5)

Alternate Protocol 3: Infection of Nonadherent Cells by Cocultivation

  Materials
  • Transfected packaging cells (see protocol 1) in 60‐mm tissue culture dishes
  • Retroviral supernatant
  • 4 mg/ml polybrene in PBS ( appendix 22), filtered and stored at 4° or −20°C
  • 105 to 106 cells/ml nonadherent target cells
  • Target cell growth medium
  • 15‐ml conical centrifuge tubes
  • 60‐mm tissue culture dishes
  • Sorvall RT‐3000B centrifuge (or equivalent)
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F) and for assaying of reporter gene expression (e.g., units 9.6 9.8 & 9.10) or drug selection (unit 9.5)

Alternate Protocol 4: Infection of Nonadherent Cells by Spin Infection

  Materials
  • Retroviral supernatant
  • 4 mg/ml polybrene in PBS ( appendix 22), filtered and stored at 4° or −20°C
  • Target cell growth medium
  • Exponentially growing nonadherent target cells
  • 24‐well tissue culture plates
  • Sorvall RT‐3000B centrifuge with microplate carrier (or equivalent)
  • 10‐cm tissue culture dishes
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F) and for assaying of reporter gene expression (e.g., units 9.6 9.8 & 9.10) or drug selection (unit 9.5)
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Figures

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Literature Cited

Literature Cited
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