Elsevier

Methods

Volume 19, Issue 3, November 1999, Pages 386-393
Methods

Regular Article
Using Inducible Vectors to Study Intracellular Trafficking of GFP-Tagged Steroid/Nuclear Receptors in Living Cells

https://doi.org/10.1006/meth.1999.0874Get rights and content

Abstract

Intracellular trafficking and localization of proteins can now be efficiently visualized by fusion of a polypeptide to the green fluorescent protein (GFP). Many spectral variants of this reagent are now available, providing powerful tools for studies in living cells. This approach is particularly useful for members of the steroid/nuclear receptor superfamily, since these molecules frequently undergo rapid subcellular redistribution on ligand activation. A major roadblock in the application of this technology concerns problems associated with transient transfections. This technique produces cell populations that are highly heterogeneous with respect to the newly introduced protein and usually contain the protein in a highly overexpressed state. In addition, long-term studies related to cell cycle and cellular differentiation are essentially impossible with this approach. These problems can be overcome by introduction of the GFP fusion into cells under appropriate induction control. We describe application of the tetracycline regulatory system to inducible control of a glucocorticoid receptor (GR)/GFP chimera. Intracellular concentrations of GFP–GR can be very effectively controlled in this system, providing an ideal environment in which to study subcellular trafficking of the receptor and interactions with a variety of intracellular targets.

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Cited by (0)

1

Current address: Department of Obstetrics and Gynecology and Department of Molecular and Medical Pharmacology, 27-139 CHS, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1740.

2

To whom correspondence should be addressed. Fax: (301) 496-4951. E-mail: [email protected].

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