New adenovirus vectors for protein production and gene transfer (original) (raw)

Abstract

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression.

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References

• Acsadi G, Jani A, Massie B, Simoneau M, Holland P, Blaschuk K and Karpati G (1994) A differential efficiency of adenovirus-mediated _in vivo_gene transfer into skeletal muscle cells of different maturity. Hum Mol Genetic 3: 579-584.
  CAS Google Scholar
• Acsadi G, Massie B and Jani A (1995) Adenovirus-mediated gene transfer into striated muscles. J Mol Med 73: 165-180.
  Article CAS Google Scholar
• Berkner KL (1988) Development of adenovirus vector for the expression of heterologous genes. BioTechniques 6: 616-629.
  CAS Google Scholar
• Berkner KL (1992) Expression of heterologous sequences in adenoviral vectors. Curr Top Microbiol Immunol 158: 39-66.
  CAS Google Scholar
• Dayan A, Bertrand R, Beauchemin M, Chahla D, Mamo A, Filion M, Skup D, Massie B and Jolivet J (1995) Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetaseencoding cDNA. Gene 165: 307-311.
  Article CAS Google Scholar
• Garnier A, Côté J, Nadeau I, Kamen A and Massie B (1994) Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells. Cytotechnology 15: 145-155.
  Article CAS Google Scholar
• Gossen M and Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547-5551.
  Article CAS Google Scholar
• Gossen M, Freundlieb S, Bender G, Müller G, Hillen W and Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268: 1766-1769.
  CAS Google Scholar
• Graham FL and Prevec L (1992) Adenovirus-based expression vectors and recombinant vaccines, In RW Ellis (Ed.) Vaccines: New Approaches to Immunological Problems. Butterworth-Heinemann, Boston, Mass, pp. 363-390.
  Google Scholar
• Heim R, Cubitt AB and Tsien RY (1995) Improved green fluorescence. Nature 373: 663-664.
  Article CAS Google Scholar
• Hu SX, Ji W, Zhou Y, Logothetis C and Xu HJ (1997) Development of an adenovirus vector with tetracycline-regulatable human tumor necrosis factor alpha gene expression. Cancer Res 57: 3339-3343.
  CAS Google Scholar
• Jani A, Lochmüller H, Acsadi G, Simoneau M, Huard J, Garnier A, Karpati G and Massie B (1997) Generation, validation and large scale production of adenoviral recombinants with large inserts such as 6.3 kb human dystrophin cDNA. J Virolog Meth 64: 111-124.
  Article CAS Google Scholar
• Lamarche N, Massie B, Richer M, Paradis H and Langelier Y (1990) High level expression in 293 cells of the herpes simplex virus type 2 ribonucleotide reductase subunit 2 using an adenovirus vector. J Gen Virol 71: 1785-1792.
  CAS Google Scholar
• Massie B (1998) BMAdE1, a new complementing cell line for the large scale production of E1-deleted Adenovirus (Ad) vectors devoid of RCA (replication competent Ad). filed on 26/04/96; Issued 09/98).
• Massie B, Couture F, Lamoureux L, Mosser DD, Guilbault C, Jolicoeur P, Belanger F and Langelier Y (1998) Inducible over-expression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette. J Virol 72: 2289-2296.
  CAS Google Scholar
• Massie B, Dionne J, Lamarche N, Fleurent J and Langelier Y (1995) Improved adenovirus vector provides herpes simplex virus ribonucleotide reductase R1 and R2 subunits very efficiently. Bio/Technology 13: 602-608.
  Article CAS Google Scholar
• McKinnon RD, Bacchetti S and Graham FL (1982) Tn5 mutagenensis of the transforming genes of human adenovirus type 5. Gene 19: 33-42.
  Article CAS Google Scholar
• Mittereder N, March K and Trapnell BC (1996) Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 70: 7498-7509.
  CAS Google Scholar
• Mosser DD, Caron AW, Bourget L, Jolicoeur P and Massie B (1997a) Use of a dicistronic expression cassette encoding the green-fluorescent protein for the screening and selection of cells expressing inducible gene products. Biotechniques 22: 150-161.
  CAS Google Scholar
• Mosser DD, Caron AW, Bourget L, Denis-Larose C and Massie B (1997b) Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol 17: 5317-5327.
  CAS Google Scholar
• Palm GJ, Zdanov A, Gaitanaris GA, Stauber R, Pavlakis GN and Wlodawer A (1997) The structural basis for spectral variations in green fluorescent protein. Nat Struct Biol 4: 361-365.
  Article CAS Google Scholar
• Rao L, Modha D and White E (1997) The E1B 19K protein associates with lamins _in vivo_and its proper localization is reuired for inhibition of apoptosis. Oncogene 15: 1587-1597.
  Article CAS Google Scholar
• Stauber RH, Ilorie K, Carney P, Hudson EA, Tarasova NI, Galtanaris GA and Pavlakis GN (1998) Development and applications of enhanced green fluorescent protein mutants. BioTechniques 24: 462-471.
  CAS Google Scholar
• Stratford-Perricaudet L D and Perricaudet M (1994) Gene therapy: the advent of adenovirus, In John A Wolff (Ed.) Gene Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser Boston, Cambridge, Mass, pp. 344-362.
  Google Scholar
• Teodoro JG and Branton PE (1997) Regulation of apoptosis by viral gene products. J Virol 71: 1739-1746.
  CAS Google Scholar
• Trapnell BC and Gorzilia M (1994) Gene therapy using adenoviral vectors. Curr Opinion in Biotech 5: 617-625.
  Article CAS Google Scholar
• White E, Cipriani R, Sabbatini P and Denton A (1991) Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. Virol 65: 2968-2978.
  CAS Google Scholar
• Xu ZZ, Krougliak V, Prevec L, Graham FL and Both GW (1995) Investigation of promoter function and animal cells infected with human recombinant adenoviruses expressing rotavirus antigen VP7sc. J Gen Virol 76: 1971-1980.
  Article CAS Google Scholar

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Author notes

1. Isabelle Vitté-mony
   Present address: Diagnocure Inc., Québec, Québec, Canada
2. France Couture
   Present address: Centre de Recherche Hospitalier de l'Université Laval, Québec, Québec, Canada
3. Luc Paquet
   Present address: Exogen Neuroscience Inc., Montré al, Québec, Canada
4. Pierre Jolicoeur
   Present address: Biophage Inc., 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada

Authors and Affiliations

1. Institut de Recherches en Biotechnologie, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada
   Bernard Massie, Dick D. Mosser, Maria Koutroumanis, Isabelle Vitté-mony, Linda Lamoureux, France Couture, Luc Paquet, Claire Guilbault, Julie Dionne, Dounia Chahla & Pierre Jolicoeur
2. Département de Microbiologie et Immunologie, Université de Montréal, Montréal, Québec, Canada, H3C 3J7
   Bernard Massie
3. Institut du Cancer de Montréal, Centre de Recherche Louis Charles Simard, 1560 Sherbrooke Est, Montréal, Québec, H2L 4M1, Canada
   Claire Guilbault & Yves Langelier

Authors

1. Bernard Massie
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2. Dick D. Mosser
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3. Maria Koutroumanis
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4. Isabelle Vitté-mony
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5. Linda Lamoureux
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6. France Couture
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7. Luc Paquet
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8. Claire Guilbault
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9. Julie Dionne
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10. Dounia Chahla
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11. Pierre Jolicoeur
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12. Yves Langelier
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Corresponding author

Correspondence toBernard Massie.

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Massie, B., Mosser, D.D., Koutroumanis, M. et al. New adenovirus vectors for protein production and gene transfer.Cytotechnology 28, 53–64 (1998). https://doi.org/10.1023/A:1008013211222

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• Issue Date: November 1998
• DOI: https://doi.org/10.1023/A:1008013211222