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Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

In Vitro Cellular & Developmental Biology - Animal volume 33, pages 575–579 (1997)Cite this article

Summary

We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.

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References

  • Artelt, P.; Grannemann, R.; Stocking, C., et al. The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells. Gene 99:249–254; 1991.

    PubMed  Article  CAS  Google Scholar 

  • Birnbaum, M. J.; Clem, R. J.; Miller, L. K. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with cys/his sequence motifs. J. Virol. 68:2521–2528; 1994.

    PubMed  CAS  Google Scholar 

  • Cartier, J. L.; Hershberger, P. A.; Friesen, P. D. Suppression of apoptosis in insect cells stably transfected with baculovirus p35: dominant interference by N-terminal sequences p351–76. J. Virol. 68:7728–7737; 1994.

    PubMed  CAS  Google Scholar 

  • Clem, R. J.; Miller, L. K. Control of programmed cell death by the baculovirus genes p35 and iap. Mol. Cell. Biol. 14:5212–5222; 1994.

    PubMed  CAS  Google Scholar 

  • de la Luna, S.; Soria, I.; Pulido, D., et al. Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker. Gene 62:121–126; 1988.

    PubMed  Article  Google Scholar 

  • Gorman, C. M.; Moffat, L. F.; Howard, B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044–1051; 1982.

    PubMed  CAS  Google Scholar 

  • Henderson, J.; Atkinson, A. E.; Lazarus, C. M., et al. Stable expression of maize auxin-binding protein in insect cell lines. FEBS Lett. 371:293–296; 1995.

    PubMed  Article  CAS  Google Scholar 

  • Jarvis, D. L. Effects of baculovirus infection on IE-1 mediated foreign gene expression in stably transformed insect cells. J. Virol. 67:2583–2591; 1993.

    PubMed  CAS  Google Scholar 

  • Jarvis, D. L.; Fleming, J. G. W.; Kovacs, G. R., et al. Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Bio/Technology 8:950–955; 1990.

    PubMed  Article  CAS  Google Scholar 

  • Jarvis, D. L.; Guarino, L. A. Continuous foreign gene expression in transformed lepidopteran insect cells. In: Richardson, C. D., ed. Baculovirus expression protocols. Humana Press; Totowa, NJ: 39:187–202; 1995.

    Chapter  Google Scholar 

  • Lahoz, E. G.; Lopez de Haro, M. S.; Esponda, P., et al. Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under the control of the rabbit uteroglobin promoter in transgenic mice. Gene 117:255–258; 1992.

    Article  Google Scholar 

  • Lahoz, E. G.; Lopez de Haro, M. S.; Nieto, A., et al. Use of puromycin N-acetyltransferase (PAC) as a new reporter gene in transgenic animals. Nucleic Acids Res. 10:3465; 1991.

    Article  Google Scholar 

  • Levy, D. N.; Fernandes, L. S.; Williams, W. V., et al. Induction of cell differentiation by human immunodeficiency virus I vpr. Cell 72:541–550; 1993.

    PubMed  Article  CAS  Google Scholar 

  • Lu, A.; Miller, L. K. The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J. Virol. 69:975–982; 1995.

    PubMed  CAS  Google Scholar 

  • McLachlin, J. R.; Miller, L. K. Identification and characterization of vlf-1, a baculovirus gene involved in very late gene expression. J. Virol. 68:7746–7756; 1994

    PubMed  CAS  Google Scholar 

  • Morgenstern, J. P.; Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:3587–3596; 1990.

    PubMed  Article  CAS  Google Scholar 

  • Morris, T. D.; Miller, L. K. Promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines. J. Virol. 66:7397–7405; 1992.

    PubMed  CAS  Google Scholar 

  • O’Reilly, D. R.; Miller, L. K.; Luckow, V. A. Baculovirus expression vectors: a laboratory manual. New York: W. H. Freeman and Co.; 1992.

    Google Scholar 

  • Rastinejad, F.; Conboy, M. J.; Rando, T. A., et al. Tumor suppression by RNA from the 3′ untranslated region of α-tropomyosin. Cell 75:1107–1117; 1993.

    PubMed  Article  CAS  Google Scholar 

  • Richardson, C. D. Baculovirus expression protocols. Methods in Molecular Biology. v. 39. Humana Press, Totowa, New Jersey; 1995.

    Book  Google Scholar 

  • Shuler, M. L.; Wood, H. A.; Granados, R. R., et al. Baculovirus expression systems and biopesticides. New York: John Wiley and Sons, Inc.; 1995.

    Google Scholar 

  • Todd, J. W.; Passarelli, A. L.; Miller, L. K. Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69:968–974; 1995.

    PubMed  CAS  Google Scholar 

  • Vara, J. A.; Portela, A.; Ortin, J., et al. Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucleic Acids Res. 14:4617–4624; 1986.

    PubMed  Article  CAS  Google Scholar 

  • Vaughn, J. L.; Goodwin, R. H.; Thompkins, G. J., et al. Establishment of two insect cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13:213–217; 1977.

    PubMed  Article  CAS  Google Scholar 

  • Vulsteke, V.; Janssen, I.; Vanden Broeck, J., et al. Baculovirus immediate early gene promoter based expression vectors for transient and stable transformation of insect cells. Insect Mol. Biol. 2:195–204; 1993.

    PubMed  CAS  Google Scholar 

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Affiliations

  1. Department of Genetics, University of Georgia, 30602, Athens, Georgia

    Lois K. Miller

  2. Department of Entomology, The University of Georgia, 413 Biological Sciences Building, 30602, Athens, Georgia

    Jeanne R. McLachlin & Lois K. Miller

Authors
  1. Jeanne R. McLachlin
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  2. Lois K. Miller
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McLachlin, J.R., Miller, L.K. Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis. In Vitro Cell.Dev.Biol.-Animal 33, 575–579 (1997). https://doi.org/10.1007/s11626-997-0101-7

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  • DOI: https://doi.org/10.1007/s11626-997-0101-7

Key words

  • Spodoptera frugiperda cells
  • puromycin acetyltransferase
  • Drosophila hsp70 promoter
  • dominant selectable marker
  • apoptosis