An adenoviral vector system for functional identification of nuclear receptor ligands.
A recombinant adenovirus system has been designed that confers glucocorticoid responsiveness upon infected cells in culture. Two mutually dependent viruses are required: a trans-activator virus containing the human glucocorticoid receptor transcription unit and a second receptor virus harboring a glucocorticoid response element linked to the firefly luciferase gene. Another reciprocal pair of viruses has been generated; one member expresses the rat thyroid hormone receptor alpha, while the other contains the luciferase gene regulated by a thyroid hormone-responsive DNA element. Corticosteroid- or thyroid hormone-induced transcription can be efficiently and accurately quantitated from cells coinfected with the appropriate complementary virus pair 20 h after infection in 96-well microtiter plates. This coinfection assay offers a convenient way to measure transcriptional activation by nuclear receptors and has certain key advantages over the commonly used cotransfection method. Its sensitivity and precision make it a practical approach to rapidly identify substances extracted from complex biological samples activating candidate "orphan" nuclear receptor molecules.
Shih
W
W
Ligand Pharmaceuticals, Inc., San Diego, CA 92121.
Mears
T
T
Bradley
D J
DJ
Parandoosh
Z
Z
Weinberger
C
C
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010957:Plasmids; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014222:Triamcinolone Acetonide / Q000378:metabolism / Q000494:pharmacology
D004274:DNA, Recombinant; D011956:Receptors, Cell Surface; D011965:Receptors, Glucocorticoid; D011988:Receptors, Thyroid Hormone; D003907:Dexamethasone; D008156:Luciferases; D014222:Triamcinolone Acetonide