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Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells

Articles Biotechnology

Summary

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adhrenent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven, expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (β-actin) promoters fused to luciferase or β-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that β-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous β-actin promoter in suspension was consistent with the response of the endogenous β-actin promoter activity because the steady-state level of β-actin messenger riboncleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.

Key words

over-agar soft agar transient transfection human β-actin promoter JB6 cells 

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References

  1. Assoian, R. K.; Boardman, L. A.; Drosinos, S. A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA. Anal. Biochem. 177:95–99; 1989.PubMedCrossRefGoogle Scholar
  2. Brinster, R. L.; Chen, H. Y.; Trumbauer, M.; Senear A. W.; Warren, R.; Palmiter, R. D. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27:223–231; 1981.PubMedCrossRefGoogle Scholar
  3. Chang, P. L.; Lee, T. F.; Garretson, K.; Prince, C. W.. Calcitriol enhancement of TPA-induced tumorigenic transformation is mediated through vitamin D receptor-dependent and-independent pathways. Clin. Exp. Metastasis 15:580–592; 1997.PubMedCrossRefGoogle Scholar
  4. Chang, P. L.; Tucker, M. A.; Hicks, P. H.; Prince, C. W. Novel protein kinase C isoforms and mitogen-activated kinase mediate phorbol ester-induced osteopontin expression. Int. J. Biochem. Cell Biol. 34:1142–1151; 2002.PubMedCrossRefGoogle Scholar
  5. Chomczynski, P.; Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162:156–159; 1987.PubMedCrossRefGoogle Scholar
  6. Colburn, N. H. Tumor promoter produces anchorage independence in mouse epidermal cells by an induction mechanism. Carcinogenesis 1:951–954; 1980.PubMedCrossRefGoogle Scholar
  7. Dike, L. E.; Farmer, S. R. Cell adhesion induces expression of growth-associated genes in suspension-arrested fibroblasts. Proc. Natl. Acad. Sci. USA 85:6792–6796; 1988.PubMedCrossRefGoogle Scholar
  8. Dong, Z.; Birrer, M. J.; Watts, R. G.; Matrisian, L. M.; Colburn, N. H. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc. Natl. Acad. Sci. USA 91:609–613; 1994a.PubMedCrossRefGoogle Scholar
  9. Dong, Z.; Cmarik, J. L.; Wendel, E. J.; Colburn, N. H. Differential transformation efficiency but not AP-1 induction under anchorage-dependent and-independent conditions. Carcinogenesis 15:1001–1004; 1994b.PubMedCrossRefGoogle Scholar
  10. Eto, I. Promotion-sensitive epidermal and mammary epithelial cells maintained in suspension over agarose. Cell Prolif. 31:71–92; 1998.PubMedCrossRefGoogle Scholar
  11. Gilmour, S. K.; Verma, A. K.; Madara, T.; O'Brien, T. G. Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis. Cancer Res. 47:1221–1225; 1987.PubMedGoogle Scholar
  12. Glanville, N.; Durnam, D. M.; Palmiter, R. D. Structure of mouse metallothionein-I gene and its mRNA. Nature 292:267–269; 1981.PubMedCrossRefGoogle Scholar
  13. Goldman, L. A.; Cutrone, E. C.; Kotenko, S. V.; Krause, C. D.; Langer, J. A. Modifications of vectors pEF-BOS, pcDNA1 and pcDNA3 result in improved convenience and expression. Biotechniques 21:1013–1015; 1996.PubMedGoogle Scholar
  14. Jansen, A. P.; Colburn, N. H.; Verma, A. K. Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation. Oncogene 18:5806–5813; 1999.PubMedCrossRefGoogle Scholar
  15. McGarrity, G. J.; Steiner, T.; Vanaman, V. Detection of mycoplasma infection of cell cultures by DNA fluorochrome staining. In: Tully, J. G.; Razin, E., ed. Methods in mycoplasmology. New York: Academic Press; 1983:155–208.Google Scholar
  16. Mizushima, S.; Nagata, S. pEF-BOS, a powerful mammalian expression vecton. Nucleic Acids Res. 18:5322; 1990.PubMedCrossRefGoogle Scholar
  17. Qin, H.; Gunning, P. The 3′-end of the human beta-actin gene enhances activity of the beta-actin expression vector system: construction of improved vectors. J. Biochem. Biophys. Methods 36:63–72; 1997.PubMedCrossRefGoogle Scholar
  18. Shin, S. I.; Freedman, V. H.; Risser, R.; Pollack, R. Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl. Acad. Sci. USA 72:4435–4439; 1975.PubMedCrossRefGoogle Scholar
  19. Watt, F. M.; Kubler, M. D.; Hotchin, N. A.; Nicholson, L. J.; Adams, J. C. Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions. J. Cell Sci. 106:175–182; 1993.PubMedGoogle Scholar
  20. Young, M. R.; Li, J. J.; Rincon, M.; Flavell, R. A.; Sathyanarayana, B. K.; Hunziker, R.; Colburn, N. Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion. Proc. Natl. Acad. Sci. USA 96:9827–9832; 1999.PubMedCrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 2003

Authors and Affiliations

  1. 1.Department of Nutrition SciencesUniversity of Alabama at BirminghamBirmingham
  2. 2.Comprehensive Cancer CenterUniversity of Alabama at BirminghamBirmingham

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