Gene Expression Using the Vaccinia Virus/ T7 RNA Polymerase Hybrid System

Orna Elroy‐Stein1, Bernard Moss2

1 Tel Aviv University, Tel Aviv, null, 2 National Institute of Allergy and Infectious Diseases, Bethesda, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.15
DOI:  10.1002/0471140864.ps0515s14
Online Posting Date:  May, 2001
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Abstract

This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter (pT7). Using liposome-mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large-scale work, protocols are provided for insertion of the pT7-regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7-3 into cells grown in suspension or for direct transfection into OST7-1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse-labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Liposome-Mediated Transfection Following Recombinant Vaccinia Virus (vTF7-3) Infection
  • Basic Protocol 2: Coinfection with Two Recombinant Vaccinia Viruses
  • Basic Protocol 3: Infection of OST7-1 Cells with a Single Virus
  • Basic Protocol 4: Gene Expression using the Vote System
  • Support Protocol: Detection of Expressed Protein using Pulse Labeling
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Liposome-Mediated Transfection Following Recombinant Vaccinia Virus (vTF7-3) Infection

 Materials
  • Recombinant plasmid: gene of interest subcloned into pTF7-5 or pTM1 vectors (available from B. Moss, e-mail bmoss@nih.gov; Figs. 5.15.1 and 5.15.2) or other plasmid containing the T7 promoter (e.g., pBluescript, Stratagene)
  • Confluent CV-1 cell monolayer (ATCC #CCL70; unit 5.12)
  • Complete DMEM-10 (appendix 3C)
  • vTF7-3 vaccinia virus stock (ATCC #VR-2153)
  • Opti-MEM I reduced serum medium (Life Technologies)
  • Liposome suspension (Lipofectin or TransfectAce; Life Technologies)
  • 6-well tissue culture dishes with 35-mm-diameter wells
  • Cup sonicator (385-W)
  • 12 × 75–mm polystyrene tubes (Falcon)
  • Additional reagents and equipment for purification of plasmid (appendix 4C)

Basic Protocol 2: Coinfection with Two Recombinant Vaccinia Viruses

 Materials
  • vTF7-3 vaccinia virus stock (ATCC #VR-2153)
  • Stock of recombinant vaccinia virus encoding the gene of interest under control of pT7 (units 5.12 5.13)
  • Confluent monolayer culture of CV-1 cells (unit 5.12) or HeLa S3 cells from a spinner culture (unit 5.12)
  • 0.25 mg/ml trypsin (Worthington 2× crystalline and salt-free; filter sterilize and stored at –20°C)
  • Complete DMEM-10, MEM spinner-5, or MEM-2.5 (depends on cell type; see below and unit 5.12)
  • Sorvall H-6000 rotor or equivalent
  • 6-well tissue culture dishes with 35-mm-diameter wells or larger tissue culture flasks for scaled-up analysis
  • Additional reagents and equipment for counting cells with a hemacytometer (appendix 3C), vaccinia virus analysis (unit 5.14), and protein purification (Chapter 6)

Basic Protocol 3: Infection of OST7-1 Cells with a Single Virus

 Materials
  • Confluent monolayer of OST7-1 cells (available from B. Moss, e-mail bmoss@nih.gov)
  • Complete DMEM-10 (appendix 3C) containing 400 potent µg/ml Geneticin (G418, unit 5.10; Life Technologies; prepared from 80 mg/ml stock in PBS, filter sterilized and stored at –20°C)
  • Stock of recombinant vaccinia virus containing the gene of interest under the control of pT7 promoter (unit 5.12 & unit 5.13)
  • 0.25 mg/ml trypsin (Worthington 2× crystalline and salt-free; filter sterilize and store at –20°C)
  • Complete MEM-2.5 (unit 5.12)
  • 6-well tissue culture dish with 35-mm-diameter wells

Basic Protocol 4: Gene Expression using the Vote System

 Materials
  • Vaccinia virus vT7lacOI (available from B. Moss, e-mail bmoss@nih.gov)
  • DNA containing gene of interest
  • pVOTE.1 or pVOTE.2 (available from B. Moss)
  • CV-1 or BS-C-1 confluent monolayer (unit 5.12)
  • Cells for protein expression (e.g., HeLa)
  • Isopropyl-1-thio--d-galactoside (IPTG)
  • Additional reagents and equipment for PCR (appendix 4J), restriction enzyme digestion (appendix 4I), preparation of vaccinia virus stock (unit 5.12), infection of cells with vaccinia virus, transfection of infected cells with a vaccinia vector, harvest of vaccinia virus, selection of recombinant vaccinia virus plaques, and plaque purification of virus (unit 5.13), and determination of protein expression in the vaccinia system (unit 5.14; also see Support Protocol in this unit)

Support Protocol: Detection of Expressed Protein using Pulse Labeling

 Additional Materials
  • Infected cells expressing the desired T7-regulated gene of interest (see Basic Protocol 1, Basic Protocol 2, Basic Protocol 3, or Basic Protocol 4) in a 6-well tissue culture dish
  • Methionine- or cysteine-free, serum-free MEM (Life Technologies, Select-Amine Kit)
  • 10 mCi/ml [35S]methionine (1175 Ci/mmol; Amersham) or 15 mCi/ml [35S]cysteine (600 Ci/mmol; Amersham)
  • Phosphate-buffered saline (PBS; appendix 2E), ice-cold
  • Cell lysis buffer (unit 5.14)
  • 6× SDS sample buffer (unit 10.1)
  • Fixing solution: 50% (v/v) methanol, 10% (v/v) acetic acid, 40% H2O
  • Fluorographic solution (EN3 HANCE from Du Pont NEN; or Amplify from Amersham)
  • Cell scraper
  • 95°C water bath
  • Additional reagents and equipment for denaturing (SDS) gel electrophoresis (unit 10.1) and autoradiography (unit 10.11)
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Figures

  •  FigureFigure 5.15.1 pTF7-5 (Fuerst et al., 1986) contains pT7 and a terminator, between which is a unique BamH1 site for insertion of a gene. The derivative, pEB2 (Berger et al., 1988) contains unique EcoRI and StuI sites followed by translation stop codons in all three reading frames. The expression cassette is flanked by segments of the vaccinia virus TK gene; thus, TK– selection can be used for isolation of recombinant plaques (unit 5.13).
    View Image
  •  FigureFigure 5.15.2 pTM1 contains the encephalomyocarditis virus untranslated leader region (EMCV-UTR) downstream of pT7. The NcoI site contains the translation initiation codon and should be used for insertion of the 5¢ end of the protein-coding DNA segment. The 3¢ end of the DNA can be inserted into any downstream sites preceding the T7 terminator. The expression cassette is flanked by segments of the vaccinia virus TK gene; thus, TK– selection can be used for isolation of recombinant virus (unit 5.13). pTM3 is the same as pTM1 except it includes the guanine phosphoribosyltransferase (gpt) gene to permit mycophenolic acid selection for recombinant virus (Moss et al., 1990; unit 5.10).
    View Image
  •  FigureFigure 5.15.3 Schematic representation of the VOTE expression system. Portions of a recombinant vaccinia virus genome containing regulatory elements are diagrammed. Transcription of the lacI gene under control of a vaccinia virus early/late promoter (pE/L) results in continuous synthesis of repressor monomers (represented by solid circles) which assemble into tetramers and bind to the lacO adjacent to a vaccinia virus late (pL) promoter, preventing transcription of the bacteriophage T7 RNA polymerase gene (T7gene1), and to the modified lacO (SLO) next to the T7 promoter (pT7), preventing transcription of the target gene should leaky synthesis of the T7 RNA polymerase occur. Upon induction, the repressor is inactivated, vaccinia virus RNA polymerase transcribes T7 gene 1, and T7 RNA polymerase is made. The latter binds to the T7 promoter and transcribes the target gene from the SLO to the triple terminator (TT). Translation of the target gene mRNA is enhanced by the EMC leader. From Ward et al., 1995; reprinted with permission of the National Academy of Sciences.
    View Image

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Literature Cited

Literature Cited
    Barret, N., Mitterer, A., Mundt, W., Eibl, J., Eibl, M., Gallo, R.C., Moss, B., and Dorner, F. 1989. Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity. AIDS Res. Hum. Retroviruses 5:159-171.
    Berger, E.A., Fuerst, T.R., and Moss, B. 1988. A soluble recombinant polypeptide comprising the amino-terminal half of the extracellular region of the CD4 molecule contains an active binding site for human immunodeficiency virus. Proc. Natl. Acad. Sci. U.S.A. 85:2357-2361.
    Earl, P.L., Koenig, S., and Moss, B. 1990. Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: Analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses. J. Virol. 65:31-41.
    Elroy-Stein, O. and Moss, B. 1990. Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 87:6743-6747.
    Elroy-Stein, O., Fuerst, T.R., and Moss, B. 1989. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5¢ sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc. Natl. Acad. Sci. U.S.A. 86:6126-6130.
    Felgner, P.L., Gadek, T.R., Holm, H., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.A., and Danielsen, G.A. 1987. Lipofection: A highly efficient, lipid-mediated DNA transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417.
    Fuerst, T.R. and Moss, B. 1989. Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5¢ untranslated leader. J. Mol. Biol. 206:333-348.
    Fuerst, T.R., Niles, E.G., Studier, F.W., and Moss, B. 1986. Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 83:8122-8126.
    Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W.A., and Fuerst, T.R. 1990. New mammalian expression vectors. Nature (Lond.) 348:91-92.
    Rose, J.K., Buonocore, L., and Whitt, M.A. 1991. A new cationic liposome reagent mediating nearly quantitive transfection of animal cells. Biotechniques 10:520-525.
    Usdin, T.B., Brownstein, M.J., Moss, B., and Isaacs, S.N. 1993. SP6 RNA polymerase containing vaccinia virus for rapid expression of cloned genes in tissue culture. BioTechniques 14:222-224.
    Ward, G.A., Stover, C.K., Moss, B., and Fuerst, T.R. 1995. Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 92:6773-6777.
    Wyatt, L.S., Moss, B., and Rozenblatt, S. 1995. Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology 210:202-205.
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