Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses

Cheryl Isaac Murphy1, Helen Piwnica‐Worms2, Stefan Grünwald3, William G. Romanow4, Nicole Francis5, Hua‐Ying Fan5

1 Aquila Biopharmaceuticals, Worcester, Massachusetts, 2 Washington University School of Medicine, St. Louis, Missouri, 3 Pharmingen, San Diego, California, 4 Corning Costar, Portsmouth, New Hampshire, 5 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.10
DOI:  10.1002/0471142727.mb1610s65
Online Posting Date:  February, 2004
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Abstract

This unit describes the maintenance and care of insect cell cultures as well as the generation, purification, and storage of recombinant baculoviruses. Procedures are included for maintenance and subculturing of insect cells and cotransfection of insect cells with linearized baculovirus DNA and recombinant transfer plasmid containing the gene of interest. In the event that the linearized virus is not available, wildÔÇÉtype baculovirus (AcMNPV) DNA may be used to produce recombinant baculoviruses. A procedure is also included for the generation of recombinant baculoviruses using a novel method, direct cloning, which eliminates the need to first clone the gene of interest into a baculoviral transfer vector. Preparation of baculovirus infection stocks from both monolayer and suspension cultures is also described. Finally, a protocol is given for a plaque assay to be used for determining the titer of baculoviral stocks as well as for selection of recombinants and plaque purification.

     
 
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Table of Contents

  • Basic Protocol 1: Maintenance and Culture of Insect Cells
  • Basic Protocol 2: Cotransfection of Insect Cells Using Linearized Baculoviral DNA
  • Alternate Protocol 1: Generation of Recombinant Baculovirus by Preparation and Transfection of Bacmid DNA Using the Bac‐to‐Bac System
  • Alternate Protocol 2: Generation of Recombinant Baculovirus Using Wild‐Type Baculoviral DNA
  • Alternate Protocol 3: Generation of Recombinant Baculoviruses by Direct Cloning
  • Basic Protocol 3: Preparation of Baculovirus Stocks
  • Basic Protocol 4: Titering Baculovirus Stocks Using Plaque Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Maintenance and Culture of Insect Cells

  Materials
  • TNM‐FH insect medium (see recipe) containing 10% fetal bovine serum (FBS), with and without 20% (v/v) DMSO
  • Spodoptera frugiperda (Sf9) cells (ATCC #CRL 1711) derived from fall armyworm ovaries (also see unit 16.9)
  • 70% ethanol
  • 0.4% trypan blue stain (Life Technologies)
  • Serum‐free insect cell culture medium (BaculoGold Protein‐Free Insect Medium from Pharmingen; Sf‐900 II from Life Technologies; HyQ‐CCM3 and HyQSFX‐Insect from Hyclone; or ExCell 401 from JRH Biosciences)
  • 60‐mm tissue culture plates or 25‐cm2 flasks
  • 27°C incubator (humidification optional)
  • Spinner culture flasks (for suspension cultures) and stir plate for multiple spinner flasks (all available from Techne or Bellco) or disposable (plastic) shaker flasks (Corning) and shaking incubator set to 27°C, 90 to 150 rpm
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent)
  • Screw‐top cryostat freezing vials
  • Liquid nitrogen freezer
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3F)

Basic Protocol 2: Cotransfection of Insect Cells Using Linearized Baculoviral DNA

  Materials
  • Spodoptera frugiperda (Sf9) cells growing in tissue culture at 50% to 70% confluence or growing in suspension culture at 1–1.5 × 106 cells/ml (see protocol 1)
  • TNM‐FH insect medium (see recipe) containing 10% fetal bovine serum (FBS)
  • Linearized ORF 1629–deleted AcMNPV DNA (e.g., BaculoGold from Pharmingen; see Fig. )
  • Recombinant baculovirus transfer vector containing gene of interest (unit 16.9)
  • Transfection buffer B (see recipe)
  • Control transfer vector: pVL1392‐XylE (Pharmingen)
  • Transfection buffer A (see recipe)
  • 500 mM catechol/50 mM sodium bisulfate
  • 60‐mm tissue culture plates
  • 27°C incubator (humidification optional)
  • Inverted microscope
  • 15‐ml conical centrifuge tubes
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent), 4°C
  • Additional reagents and equipment for amplification of viral supernatants to produce baculoviral stocks (see protocol 6) and plaque assay of baculovirus (see protocol 7)

Alternate Protocol 1: Generation of Recombinant Baculovirus by Preparation and Transfection of Bacmid DNA Using the Bac‐to‐Bac System

  Materials
  • cDNA of interest (e.g., unit 5.5)
  • Bac‐to‐Bac Baculovirus Expression System (Invitrogen) including:
    • pFastBac plasmid vector and control plasmid
    • DH10Bac competent E. coli cells
    • Bac‐to‐Bac manual (available online at http://invitrogen.com/content/sfs/manuals/bactobac_man.pdf)
    • Cellfectin lipid transfection reagent
  • Selection plates for transposition (see recipe)
  • Bacmid selection medium (see recipe)
  • Sf9 cells in serum‐free medium
  • Serum‐free medium
  • 27°C incubator
  • 6‐well tissue culture plates
  • Additional reagents and equipment for cloning DNA into plasmids (unit 3.16), introduction of plasmid DNA into E. coli cells (unit 1.8), growing E. coli on solid (unit 1.3) and in liquid (unit 1.2) medium, alkaline lysis miniprep (unit 1.6), and preparation of insect cells for transfection (see protocol 2, steps to , in this unit), and amplification of recombinant baculovirus to produce stocks (see protocol 6 in this unit)

Alternate Protocol 2: Generation of Recombinant Baculovirus Using Wild‐Type Baculoviral DNA

  • Wild‐type baculovirus (Dr. Max D. Summers, see above, or Pharmingen)
  • Sucrose cushion solution (see recipe)
  • 0.1× and 1× TE buffer, pH 7.4 ( appendix 22)
  • 25% and 56% (w/v) sucrose (ultrapure) in 0.1× TE buffer (filter sterilize and store up to 1 month at 4°C)
  • Extraction buffer (see recipe)
  • 10 mg/ml proteinase K (prepare fresh)
  • 10% N‐lauroylsarcosine (sodium salt; filter sterilize and store up to 1 year at 4°C)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 100% (ice‐cold) and 70% (room temperature) ethanol
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 150‐mm tissue culture dishes
  • 50‐ml conical centrifuge tubes
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent), 4°C
  • Beckman ultracentrifuge with SW‐27 or SW‐28 rotor and SW‐41 rotor (or other ultracentrifuge with equivalent rotors), 4°C, and appropriate tubes
  • Sucrose gradient maker
  • 15‐ml polypropylene centrifuge tubes
  • 50°C water bath
  • 5‐ or 10‐ml wide‐mouth pipets
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation of DNA (unit 2.1) and quantitating DNA by absorbance spectrometry ( appendix 3D)

Alternate Protocol 3: Generation of Recombinant Baculoviruses by Direct Cloning

  • Purified vector containing gene of interest, flanked by EcoRI sites
  • Klenow fragment of DNA polymerase I (unit 3.5)
  • Reaction buffer for Klenow fragment (see recipe)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 1× TE buffer ( appendix 22)
  • vEHuni or vECuni (Pharmingen), linearized, partially filled in by Klenow fragment treatment, and containing TTA ends
  • T4 DNA ligase (unit 3.14)
  • 15°C water bath
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and phenol/chloroform extraction and ethanol precipitation of DNA (unit 2.1)

Basic Protocol 3: Preparation of Baculovirus Stocks

  Materials
  • Sf9 cells in monolayer culture (see protocol 1, step ) or suspension culture (see protocol 1, step )
  • TNM‐FH insect medium (see recipe) containing 10% fetal bovine serum (FBS)
  • Baculoviral inoculum, wild‐type (unit 16.9) or recombinant (e.g., supernatant from contransfected Sf9 cells; see protocol 2), pfu determined by plaque assay (see protocol 7)
  • 150‐mm tissue culture dishes
  • 27°C incubator (humidification optional)
  • Inverted microscope
  • Beckman GPR centrifuge with GH‐3.7 horizontal rotor (or equivalent), 4°C
  • Screw‐top cryostat freezing vials
  • Liquid nitrogen freezer
  • Spinner culture flasks (for suspension cultures) and stir plate for multiple spinner flasks (all available from Techne or Bellco) or disposable (plastic) shaker flasks (Corning) and shaking incubator set to 27°C, 90 to 150 rpm

Basic Protocol 4: Titering Baculovirus Stocks Using Plaque Assay

  Materials
  • Exponentially growing Sf9 cells (see protocol 1) in monolayer culture
  • TNM‐FH insect cell medium (see recipe) with and without 10% fetal bovine serum (FBS)
  • Baculoviral stock
  • Agarose overlay (prepare 30 min before use in step ; see recipe)
  • Trypan blue overlay (optional; see recipe)
  • 60‐mm tissue culture plates
  • 27°C incubator (humidification optional)
  • 1.5‐ml screw‐top cryostat tube
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Figures

Videos

Literature Cited

   Beames, B., Braunagel, S., Summers, M.D., and Lanford, R.E. 1991. Polyhedrin initiator codon altered to AUU yields unexpected fusion protein from a baculovirus vector. BioTechniques 11:378‐383.
   Jarvis, D.L. and Garcia, A. 1994. Long‐term stability of baculoviruses stored under various conditions. BioTechniques 16:508‐513.
   Jarvis, D.L., Summers, M.D., Garcia, A., and Bohlmeyer, D.A. 1993. Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J. Biol. Chem. 268:16754‐16762.
   Jeang, K., Giam, C., Nerenberg, M., and Khoury, G. 1987. Abundant synthesis of functional T cell leukemia virus type I p40x protein in eucaryotic cells by using a baculovirus expression vector. J. Virol. 61:708‐713.
   Lu, A. and Miller, L.K. 1996. Generation of recombinant baculoviruses by direct cloning. BioTechniques 21:63‐68.
   Maiorella, B., Inlow, D., Shauger, A., and Harano, D. 1988. Large‐scale insect cell culture for recombinant protein production. Bio/Technology 6:1406‐1410.
   Murphy, C.L., McIntire, J.R., Davis, D.R., Hodgdon, H., Seals, J.R., and Young, E. 1993. Enhanced expression, secretion, and large‐scale purification of recombinant HIV‐I gp120 in insect cells using the baculovirus egt and p67 signal peptides. Protein Expr. Purif. 4:349‐357.
   O'Reilly, D.R., Miller, L.K., and Luckow, V.A. 1992. Baculovirus Expression Vectors. W.H. Freeman, New York.
Key Reference
   O'Reilly et al., 1992. See above.
  A guide assembled to aid researchers using the baculoviral expression system, containing detailed protocols for using this system effectively.
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