High-throughput screening for ligand-induced c-fos mRNA expression by branched DNA assay in Chinese hamster ovary cells.
We have developed a generally useful screening assay for receptor agonists and antagonists in Chinese Hamster Ovary (CHO) cells. Three key features of the assay make it applicable to a broad range of receptors: (1) the use of CHO cells as host cells to overexpress receptors, (2) measurement of endogenous c-fos mRNA, which responds to a wide spectrum of stimuli, and (3) the use of branched chain DNA assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute. The combination of these features provides a powerful means to screen rapidly for peptide and small molecule ligands for a variety of receptors. CHO cells overexpressing insulin receptor were used as a test system to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.
Shyamala
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V
Department of Molecular Biology, Chiron Corporation, 4560 Horton Street, Emeryville, California, 94608, USA. shyamala@cc.chiron.com
Khoja
H
H
Anderson
M L
ML
Wang
J X
JX
Cen
H
H
Kavanaugh
W M
WM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D016762:Genes, fos; D005821:Genetic Techniques; D007328:Insulin / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis* / Q000187:drug effects; D011933:Reagent Kits, Diagnostic; D011972:Receptor, Insulin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013997:Time Factors
D007328:Insulin; D009841:Oligonucleotides; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases