COS‐1 Cells as Packaging Host for Production of Lentiviruses

Crystal J. MacKenzie1, Toshi Shioda1

1 Massachusetts General Hospital Center for Cancer Research, Charlestown, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.7
DOI:  10.1002/0471143030.cb2607s50
Online Posting Date:  March, 2011
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Abstract

We present a protocol for in vitro production of recombinant lentiviruses using COS‐1 African green monkey kidney epithelial cells and HEK293T human embryonic kidney epithelial cells as packaging cells. COS‐1 and HEK293T express SV40 large T antigen, amplifying transfected circular plasmids harboring SV40 replication origin. Support protocols for evaluation of transfection efficiency by in situ β‐galactosidase enzyme activity assay and titer of infection‐capable virions are also provided. Advantages of using COS‐1 packaging cells over the standard HEK293T cells for contamination‐sensitive applications or automated processing are discussed. Curr. Protoc. Cell Biol. 50:26.7.1‐26.7.15. © 2011 by John Wiley & Sons, Inc.

Keywords: lentivirus; COS‐1; packaging; HEK293T

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Lentivirus Virions by Transfection of COS‐1 Cells or HEK293T Cells
  • Support Protocol 1: Determination of Transfection Efficiency
  • Support Protocol 2: Determination of Lentivirus Titer by Drug Selection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Lentivirus Virions by Transfection of COS‐1 Cells or HEK293T Cells

  Materials
  • COS‐1 cells (ATCC CRL‐1650) or HEK293T cells (Broad Institute); commercially available HEK293T derivatives include HEK293T/17 cells (ATCC CRL‐11268) and HEK293FT cells (Invitrogen)
  • Alcohol pads
  • Low‐FBS DMEM (see recipe)
  • 1× trypsin/EDTA (Mediatech, cat. no. 25‐053‐Cl)
  • Dulbecco's phosphate‐buffered saline (DPBS, without Ca2+/Mg2+; Mediatech, cat. no. 21040CM)
  • Opti‐MEM I reduced serum medium (Invitrogen, cat. no. 31985‐070)
  • Lentivirus vector plasmid (e.g., pCMV‐dR8.91, Broad Institute), 0.5 µg/ml in 1× TE buffer (see appendix 2A for TE buffer)
  • TransIT‐LT1 transfection agent (Mirus, cat. no. MIR‐2306; http://www.mirusbio.com/)
  • VSV‐G expression plasmid (e.g., pMD2G, Broad Institute), 0.5 µg/ml in 1× TE buffer (see appendix 2A for TE buffer)
  • Lentiviral LTR‐containing expression cassette vector (e.g., pLKO.1, Broad Institute), 20 ng/ml in 1× TE buffer (see appendix 2A for TE buffer)
  • High‐FBS DMEM (see recipe)
  • Vesphene IISE (Fisher, cat. no. 14‐415‐11)
  • 10‐cm cell culture dishes (Corning, cat. no. 430293)
  • 37°C Incubator with 5% to 10% CO 2
  • Disposable 10‐ml syringes (Becton Dickinson, cat. no. 309604)
  • 96‐well flat‐bottom cell culture plate (Corning 3599)
  • 50‐ml centrifuge tube (Corning, cat. no. 430290)
  • 96‐well round‐bottom plate (Corning, cat. no. 3790)
  • Multichannel pipettor and 50‐ml reagent reservoir (Corning, cat. no. 4870)
  • 96‐well round‐bottom deep‐well plate, 2 ml/well (Fisher, cat. no. 12‐566‐121)
  • Microamp seal (ABI, cat. no. 4306311)
  • TempPlate Sealing Foil (sterilized aluminum sealing foil; USA Scientific, cat. no. 2923‐0110)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All cell culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Determination of Transfection Efficiency

  • pSV‐β‐galactosidase (Promega, cat. no. E1081) or pCMVβ (Clontech, cat. no. 631719)
  • Glutaraldehyde fixative (see recipe)
  • Xgal substrate solution (see recipe)
  • 70% (w/w) glycerol

Support Protocol 2: Determination of Lentivirus Titer by Drug Selection

  • MCF‐7 human breast cancer cells (ATCC, HTB‐22)
  • 8 mg/ml polybrene (hexadimethrine bromide; Sigma, cat. no. H9268); filter sterilize with 0.22‐µm syringe filter and store in small aliquots up to 3 months at –20°C (avoid repeated freeze‐thaw cycles)
  • 2 mg/ml stock solution of puromycin dihydrochloride (cell‐culture grade; Sigma, cat. no. P8833); filter sterilize with 0.22‐µm syringe filter and store in small aliquots up to 3 months at –20°C (avoid repeated freeze‐thaw cycles)
  • Formaldehyde fixative (see recipe)
  • 70% ethanol
  • Crystal violet staining solution (see recipe)
  • Swinging‐bucket centrifuge with microtiter plate carrier approved for work with hazardous materials
  • Aerosol‐barrier pipet tips
  • 37°C, 5 to 10% CO 2 incubator designated for experiments with live lentivirus
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Figures

Videos

Literature Cited

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