Summary
The relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV ≈ CMV ≈ SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Brash, D. E.; Reddel, R. R.; Quanrud, M., et al. Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells. Mol. Cell. Biol. 7:2031–2034; 1987.
deWet, J. R.; Wood, K. V.; DeLuca, M., et al. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7:725–737; 1987.
Foecking, M. K.; Hofstetter, H. Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45:101–105; 1986.
Ginot, F.; Decaux, J.-F.; Cognet, M., et al. Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression. Eur. J. Biochem. 180:289–294; 1989.
Gorman, C. M.; Merlino, G. T.; Willingham, M. C., et al. The rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc. Natl. Acad. Sci. USA 79:6777–6781; 1982.
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.
Gould, M. N.; Zhang, R. Genetic regulation of carcinogenesis in the rat by susceptibility and suppressor genes. Environ. Health Perspect. 93:161–167; 1991.
Kirkland, W. L.; Yang, N.-S.; Jorgensen, T., et al. Growth of normal and malignant mammary epithelial cells in culture. JNCI 63:29–41; 1979.
Kriegler, M. Gene transfer and expression: a laboratory manual. New York: Stockton Press; 1990:96–101.
MacGregor, G. R.; Mogg, A. E.; Burke, J. F., et al. Histochemical staining of clonal mammalian cell lines expressingE. coli β-galactosidase indicates heterogenous expression of the bacterial gene. Somatic Cell Mol. Genet. 13:253–265; 1987.
MacGregor, G. R.; Caskey, C. T. Construction of plasmids that expressE. coli β-galactosidase in mammalian cells. Nucleic Acids Res. 17:2365; 1989.
Ponder, K. P.; Dunbar, R. P.; Wilson, D. R., et al. Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection. Hum. Gene Ther. 2:41–52; 1991.
Ross, S. R.; Hsu, C.-L. L.; Choi, Y., et al. Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice. Mol. Cell. Biol. 10:5822–5829; 1990.
Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989:1.21–1.84.
Wang, B.; Kennan, W. S.; Yasukawa-Barnes, J., et al. Carcinoma induction following directin situ transfer of v-Ha-ras into rat mammary epithelial cells using replication-defective retrovirus vectors. Cancer Res. 51:2642–2648; 1991.
Xian-jun, F.; Keating, A.; deVilliers, J., et al. Tissue-specific activity of heterologous viral promoters in primary rat hepatocytes and hep G2 cells. Hepatology 10:781–787; 1989.
Yang, N.-S.; Burkholder, J.; Roberts, B., et al.In Vivo andin vitro gene transfer to mammalian somatic cells by particle bombardment. Proc. Natl. Acad. Sci. USA 87:9568–9572; 1990.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Thompson, T.A., Gould, M.N., Burkholder, J.K. et al. Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer. In Vitro Cell Dev Biol - Animal 29, 165–170 (1993). https://doi.org/10.1007/BF02630949
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02630949