Abstract
Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and β-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor α (ERα)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin increases ERα promoter activity and increases functional ER activity, suggesting a reciprocal regulation between activin and estrogen signaling in the ovary. We also discuss more broadly some key considerations in the use of reporter genes and cell-based transfection assays in endocrine research.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M., Darnell, J.E. Jr. (1981) Transcriptional control in the production of liver-specific mRNAs. Cell 23:731–739.
Greenberg, M.E., Ziff, E.B. (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311:433–438.
Weaver, R.F. (1999) Molecular Biology. 1st ed: McGraw-Hill Companies, Inc.
Hu, Z.W., Hoffman, B.B. (2000) Nuclear run-on assays for measurement of adrenergic receptor transcription rate. Methods in Molecular Biology 126:169–180.
Eick, D., Kohlhuber, F., Wolf, D.A., Strobl, L.J. (1994) Activation of pausing RNA polymerases by nuclear run-on experiments. Analytical Biochemistry 218:347–351.
Sandoval, J., Rodriguez, J.L., Tur, G., et al. (2004) RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription. Nucleic Acids Research 32:e88.
Lipson, K.E., Baserga, R. (1989) Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe. Proceedings of the National Academy of Sciences of the United States of America 86:9774–9777.
Prendergast, F.G., Mann, K.G. (1978) Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskalea. Biochemistry 17:3448–3453.
Tsien, R.Y. (1998) The green fluorescent protein. Annual Review of Biochemistry 67:509–544.
Ow, D.W., DE Wet J.R., Helinski, D.R., Howell, S.H., Wood, K.V., Deluca, M. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856–859.
de Wet JR, Wood, K.V., DeLuca, M., Helinski, D.R., Subramani, S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Molecular and Cellular Biology 7:725–737.
Joung, J.K., Ramm, E.I., Pabo, C.O. (2000) A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. Proceedings of the National Academy of Sciences of the United States of America 97:7382–7387.
Jain, V.K., Magrath, I.T. (1991) A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-dgalactosidase in lacZ-transfected cells. Analytical Biochemistry 199:119–124.
Bronstein, I., Fortin, J., Stanley, P.E., Stewart, G.S., Kricka, L.J. (1994) Chemiluminescent and bioluminescent reporter gene assays. Analytical Biochemistry 219:169–181.
Bronstein, I., Fortin, J.J., Voyta, J.C., et al. (1994) Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products. BioTechniques 17:172–174, 6–7.
Bronstein, I., Martin, C.S., Fortin, J.J., Olesen, C.E., Voyta, J.C. (1996) Chemiluminescence: sensitive detection technology for reporter gene assays. Clinical Chemistry 42:1542–1546.
Kingston, R.E., Sheen, J., Moore, D. (2001) Isotopic assays for reporter gene activity. Current protocols in molecular biology, edited by Frederick M Ausubel et al. Chapter 9:Unit9 7A.
Thompson, J.F., Hayes, L.S., Lloyd, D.B. (1991) Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 103:171–177.
Carey, M., Smale, S.T. (1999) Functional Assays for Promoter Analysis. In: Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques: Cold Spring Harbor Laboratory Press, pp. 137–92.
Felgner, P.L., Gadek, T.R., Holm, M., et al. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences of the United States of America 84:7413–7417.
Zhang, K., Kurachi, S., Kurachi, K. (2003) Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene. Journal of Biological Chemistry 278:4826–4830.
Sherf, B.A. Navarroi, S.L. Hannah, R.R. and Wood, K.V. Dual-Luciferase, T.M. (1996) Reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays. Promega Notes Magazine 57:02.
Kipp, J.L., Kilen, S.M., Bristol-Gould, S., Woodruff, T.K., Mayo, K.E. (2007) Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary. Endocrinology 148:1968–1976.
Kipp, J.L., Kilen, S.M., Woodruff, T.K., Mayo, K.E. (2007) Activin regulates estrogen receptor gene expression in the mouse ovary. Journal of Biological Chemistry 282:36755–36765.
Briers, T.W., van de Voorde, A., Vanderstichele, H. (1993) Characterization of immortalized mouse granulosa cell lines. In vitro Cellular & Developmental Biology 29A:847–854.
Vanderstichele, H., Delaey, B., de Winter, J., et al. (1994) Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines. Biology of Reproduction 50:1190–1202.
Campbell, M.J. (1995) Lipofection reagents prepared by a simple ethanol injection technique. BioTechniques 18:1027–1032.
Dykema, J.C., Mayo, K.E. (1994) Two messenger ribonucleic acids encoding the common beta B-chain of inhibin and activin have distinct 5′-initiation sites and are differentially regulated in rat granulosa cells. Endocrinology 135:702–711.
Mukherjee, A., Park-Sarge, O.K., Mayo, K.E. (1996) Gonadotropins induce rapid phosphorylation of the 3′,5′-cyclic adenosine monophosphate response element binding protein in ovarian granulosa cells. Endocrinology 137:3234–3245.
Park-Sarge, O.K., Mayo, K.E. (1994) Regulation of the progesterone receptor gene by gonadotropins and cyclic adenosine 3′,5′-monophosphate in rat granulosa cells. Endocrinology 134:709–718.
Pei, L., Dodson, R., Schoderbek, W.E., Maurer, R.A., Mayo, K.E. (1991) Regulation of the alpha inhibin gene by cyclic adenosine 3′,5′-monophosphate after transfection into rat granulosa cells. Molecular Endocrinology 5:521–534.
Burkart, A.D., Mukherjee, A., Mayo, K.E. (2006) Mechanism of repression of the inhibin alpha-subunit gene by inducible 3′,5′-cyclic adenosine monophosphate early repressor. Molecular Endocrinology 20:584–597.
Campbell, K.L. (1979) Ovarian granulosa cells isolated with EGTA and hypertonic sucrose: cellular integrity and function. Biology of Reproduction 21:773–786.
Ardekani, A.M., Romanelli, J.C., Mayo, K.E. (1998) Structure of the rat inhibin and activin betaA-subunit gene and regulation in an ovarian granulosa cell line. Endocrinology 139:3271–3279.
Gehm, B.D., McAndrews, J.M., Chien, P.Y., Jameson, J.L. (1997) Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proceedings of the National Academy of Sciences of the United States of America 94:14138–14143.
Cicatiello, L., Cobellis, G., Addeo, R., et al. (1995) In vivo functional analysis of the mouse estrogen receptor gene promoter: a transgenic mouse model to study tissue-specific and developmental regulation of estrogen receptor gene transcription. Molecular Endocrinology 9:1077–1090.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kipp, J.L., Mayo, K.E. (2009). Use of Reporter Genes to Study the Activity of Promoters in Ovarian Granulosa Cells. In: Park-Sarge, OK., Curry, T. (eds) Molecular Endocrinology. Methods in Molecular Biology, vol 590. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-378-7_11
Download citation
DOI: https://doi.org/10.1007/978-1-60327-378-7_11
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-377-0
Online ISBN: 978-1-60327-378-7
eBook Packages: Springer Protocols