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Methods. 1999 Nov;19(3):386-93.

Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells.

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1
Laboratory of Receptor Biology and Gene Expression, Building 41, Room B602, National Cancer Institute, Bethesda, Maryland 20892-5055, USA.

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.

PMID:
10579933
DOI:
10.1006/meth.1999.0874
[Indexed for MEDLINE]

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