Retroviral Vector Production

A. Dusty Miller1

1 Fred Hutchinson Cancer Research Center and Department of Pathology, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.5
DOI:  10.1002/0471142905.hg1205s80
Online Posting Date:  February, 2014
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Abstract

In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication‐competent virus. Additional protocols detail plasmid transfection, virus titration, assay for replication‐competent virus, and histochemical staining to detect transfer of a vector encoding alkaline phosphatase. Curr. Protoc. Hum. Genet. 80:12.5.1‐12.5.22. © 2014 by John Wiley & Sons, Inc.

Keywords: gene therapy; retroviral vectors; retrovirus packaging cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Stable Packaging Cell Lines that Produce Vectors Carrying Selectable Markers
  • Alternate Protocol 1: Generation of Stable Packaging Cell Lines that Produce Vectors without Selectable Markers
  • Alternate Protocol 2: Production of Vector by Transient Transfection
  • Support Protocol 1: Construction of Retroviral Vectors
  • Support Protocol 2: Calcium Phosphate–Mediated Transfection of Cultured Cells
  • Support Protocol 3: Assay to Titer Vectors Carrying Selectable Markers
  • Support Protocol 4: Marker Rescue Assay for Helper Virus
  • Support Protocol 5: Staining Cultured Cells for Alkaline Phosphatase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of Stable Packaging Cell Lines that Produce Vectors Carrying Selectable Markers

  Materials
  • Two retrovirus packaging cell lines, in this example, PE501 and PT67 (see Table 12.5.1). PE501 cells are available from the author, and PT67 cells are available from the American Type Culture Collection (ATCC, http://www.atcc.org) cell line no. CRL‐12284, or from Clontech Labs (http://www.clontech.com) cat. no. 631510. Other ecotropic packaging cell lines can be substituted for the PE501 cells, including GP+E‐86 cells (Markowitz et al., ), which are available as ATCC cell line no. CRL‐9642.
  • Dulbecco's modified Eagle medium containing 4.5 g/liter glucose and 10% (v/v) FBS (DMEM/10% FBS; see )
  • Retroviral vector plasmid containing selectable marker (see protocol 4)
  • 4 mg/ml Polybrene (Sigma) in PBS ( )
  • Drug appropriate for the selectable marker carried by the vector: e.g., 0.75 mg/ml G418 (active concentration), 4 mM L‐histidinol, or 0.4 mg/ml hygromycin B
  • Cell staining solution (see recipe)
  • 6‐ and 10‐cm tissue culture dishes
  • 10‐ml syringes
  • 0.45‐µm‐pore‐size low‐protein‐binding cellulose acetate syringe filters
  • Cloning rings (unit )
  • Additional reagents and equipment for culturing and freezing mammalian cells ( ), calcium phosphate–mediated transfection (see protocol 5), isolating clones using cloning rings (unit ), assaying vector titer (see protocol 6), preparing genomic DNA (e.g., ), Southern blot hybridization (unit ), and performing marker rescue assay for helper virus (see protocol 7)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Generation of Stable Packaging Cell Lines that Produce Vectors without Selectable Markers

  Materials
  • Retrovirus packaging cells, in this example, PT67 cells (see Table 12.5.1)
  • Dulbecco's modified Eagle medium containing 4.5 g/liter glucose and 10% (v/v) FBS (DMEM/10% FBS; see )
  • Retroviral vector plasmid DNA (see protocol 4)
  • Plasmid DNA with selectable marker: e.g., pSV2neo
  • 0.75 mg/ml G418 (active concentration) or other selective drug appropriate for the plasmid with selectable marker
  • 6‐cm tissue culture dishes
  • Cloning rings (unit ), sterile
  • Additional reagents and equipment for culturing and freezing mammalian cells ( ), calcium phosphate–mediated transfection of cells (see protocol 5), isolating clones using cloning rings (unit ), and analyzing clones (see protocol 1, steps 14 to 18, and unit )
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Production of Vector by Transient Transfection

  Materials
  • Retrovirus packaging cells (see Table 12.5.1) and appropriate tissue culture medium
  • Retroviral vector plasmid DNA (see protocol 4)
  • 6‐cm tissue culture plates
  • 10‐ml syringes
  • 0.45‐µm‐pore‐size low‐protein‐binding cellulose acetate syringe filters
  • Additional reagents and equipment for culture of mammalian cells ( ) and calcium phosphate–mediated transfection (see protocol 5)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Support Protocol 1: Construction of Retroviral Vectors

  Materials
  • cDNA of interest
  • Retroviral vector in a bacterial plasmid
  • Additional reagents and equipment for constructing a plasmid (e.g., Struhl, ), transforming E. coli cells (e.g., Seidman et al., ), and preparing DNA minipreps (unit or, e.g., Engebrecht et al., )

Support Protocol 2: Calcium Phosphate–Mediated Transfection of Cultured Cells

  Materials
  • Retroviral vector plasmid DNA (see protocol 4)
  • 10 mM Tris·Cl, pH 7.5 ( )
  • Retrovirus packaging cells (see Table 12.5.1)
  • Dulbecco's modified Eagle medium containing 4.5 g/liter glucose and 10% (v/v) FBS (DMEM/10% FBS; see )
  • 2.0 M CaCl 2
  • 500 mM HEPES, pH 7.1
  • 2.0 M NaCl
  • 150 mM sodium phosphate buffer, pH 7.0 ( )
  • Sterile H 2O
  • 12 × 75–mm clear polystyrene tubes (Falcon)
  • Additional reagents and equipment for selection or screening of transfected cells (see protocol 1Basic Protocol or Alternate Protocol protocol 21 or protocol 32)
NOTE: All solutions should be sterilized by filtration through 0.22‐µm‐pore‐size sterile filters; if the plasmid DNA is not already sterile because it was ethanol precipitated during preparation, it should also be filtered.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All mammalian cell culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Support Protocol 3: Assay to Titer Vectors Carrying Selectable Markers

  Materials
  • Target cells susceptible to the vector pseudotype to be tested (e.g., NIH 3T3 or HeLa cells) and appropriate tissue culture medium
  • 4 mg/ml Polybrene (Sigma)
  • Retroviral stock to be tested (e.g., from protocol 1Basic Protocol or Alternate Protocol protocol 21 or protocol 32)
  • Selective drug: 0.75 mg/ml G418 (active concentration), 0.4 mM hygromycin B, or 4 mM L‐histidinol
  • Cell staining solution (see recipe)
  • 6‐cm tissue culture dishes
  • Additional reagents and equipment for culture and passaging of mammalian cells ( )
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Support Protocol 4: Marker Rescue Assay for Helper Virus

  Materials
  • LAPSN vector plasmid or virus (see Fig. )
  • Naïve target cells that are susceptible to the pseudotype of the retrovirus vector stock to be tested, and that do not express high levels of endogenous heat‐stable AP: e.g., NIH 3T3 or HT‐1080 cells
  • Retrovirus stock to be tested (see protocol 1Basic Protocol or Alternate Protocol protocol 21 or protocol 32), filtered through 0.45‐µm‐pore‐size low‐protein‐binding cellulose acetate syringe filter
  • 4 mg/ml Polybrene (Sigma) in PBS ( )
  • Positive control virus: amphotropic replication‐competent “helper” virus
  • 6‐cm tissue culture dishes
  • 10‐ml syringes
  • 0.45‐µm‐pore‐size low‐protein‐binding cellulose acetate syringe filters
  • Additional reagent and equipment for culturing and passaging mammalian cells ( ), transient or stable transfection of cells (see protocol 1Basic Protocol or Alternate Protocol protocol 21 or protocol 32), and staining for alkaline phosphatase activity (see protocol 8)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Support Protocol 5: Staining Cultured Cells for Alkaline Phosphatase Activity

  Materials
  • Cell cultures transduced with alkaline phosphatase–encoding vector (e.g., LAPSN) in 6‐cm tissue culture dishes
  • 0.25% (v/v) glutaraldehyde in PBS ( )
  • PBS ( )
  • Alkaline phosphatase staining buffer (see recipe)
  • Alkaline phosphatase staining solution (see recipe)
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Figures

Videos

Literature Cited

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