Production of Vesicular Stomatitis Virus G Glycoprotein (VSV‐G) Pseudotyped Retroviral Vectors

Hsin‐Lung Lo1, Jiing‐Kuan Yee1

1 City of Hope National Medical Center, Duarte, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.7
DOI:  10.1002/0471142905.hg1207s52
Online Posting Date:  January, 2007
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Abstract

Retrovirus pseudotype is defined as the genome of one retrovirus encapsidated by the envelope protein of a second virus. The host range of the pseudotype is that of the virus donating the envelope protein. Two procedures that use 293GP cells, which are derived from human kidney 293 cells, are described here. The first is based on the high transient transfection efficiency of 293 cells. The retroviral construct and an expression plasmid for VSV‐G are co‐transfected into 293GP cells that stably express MLV gag and pol proteins. Transiently generated virus is then harvested during consecutive days following DNA transfection. The second procedure involves stable 293GP cell lines containing the VSV‐G gene under the control of a promoter whose activity is regulated by tetracycline. Cell lines containing the retroviral vector of interest are established under noninduced conditions. Infectious virus can be harvested following the induction of VSV‐G expression in these cell lines.

Keywords: retrovirus; vector; transfection; pseudotype; VSV‐G; 293GP

     
 
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Table of Contents

  • Basic Protocol 1: VSV‐G Pseudotyped Retrovirus Production by Transient Transfection
  • Alternate Protocol 1: VSV‐G Pseudotyped Retrovirus Production from Stable Producer Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: VSV‐G Pseudotyped Retrovirus Production by Transient Transfection

  Materials
  • Retroviral vectors with 5′ U3 enhancer replaced by the immediate early (IE) gene enhancer of cytomegalovirus (CMV)
  • Maxi DNA purification kit (Qiagen)
  • Cultures of 293GP and HT1080 cells
  • Complete DMEM/10% FBS ( appendix 3G)
  • Plasmid pCMV‐G
  • TE79/10 (Tris‐buffered EDTA; see recipe)
  • 2 M CaCl 2 (see recipe)
  • 2× HeBS (see recipe)
  • Dulbecco's modified Eagle medium (DMEM), serum free
  • Fetal bovine serum (FBS; appendix 3G)
  • 40% polyethylene glycol (PEG; see recipe)
  • 4 mg/ml polybrene (see recipe)
  • 37% formaldehyde
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • 100‐mm tissue culture plate
  • 6‐ml polypropylene tube, sterile
  • 0.45 µm sterile nonpyrogenic syringe filter
  • Tabletop centrifuge: e.g., Allegra 6 (Beckman Coulter)
  • 14 × 89–mm Beckman Ultra‐Clear centrifuge tubes, sterilized overnight with UV light in a laminar flow hood
  • High‐speed ultracentrifuge: e.g., Beckman Coulter Optima L‐90k with SW28 rotor
  • 6‐well tissue culture plates
  • Fluorescence Activated Cell Sorter (FACS)
  • Additional reagents and equipment for cloning a gene of interest into a retroviral vector (unit 12.5) and trypsinization of cells ( appendix 3G)
NOTE: All reagents and equipment coming in contact with live cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All tissue culture incubations are performed in a humidified 37°C, 10% CO 2 incubator.

Alternate Protocol 1: VSV‐G Pseudotyped Retrovirus Production from Stable Producer Cells

  • Cultures of the 293GP and 293GP/G‐21 cells
  • Retroviral vector with the gene of interest and the GFP gene
  • 80 mg/ml hygromycin (see recipe) 1 mg/ml puromycin (see recipe)
  • 1 mg/ml tetracycline (see recipe)
  • 200 µM β‐estradiol (see recipe)
  • 6‐, 24‐ and 48‐well tissue culture plates
NOTE: All reagents and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All tissue culture incubations are performed in a humidified 37°C, 10% CO 2 incubator.
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Figures

Videos

Literature Cited

Literature Cited
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