title: Development of a facile method for high throughput screening with reporter gene assays.
abstract: This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.
authors.affiliation: Glaxo Wellcome Research and Development, Research Triangle Park, NC 27709, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Goetz
authors.forename: J L
authors.initials: JL
authors.lastname: Andrews
authors.forename: T R
authors.initials: TR
authors.lastname: Littleton
authors.forename: D M
authors.initials: DM
authors.lastname: Ignar
mesh_terms: D000818:Animals; D016466:CHO Cells; D002116:Calcitonin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D004790:Enzyme Induction / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D018043:Receptors, Corticotropin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044101:Receptors, Melanocortin; D015203:Reproducibility of Results; D020218:Response Elements / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors; D014162:Transfection; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D018043:Receptors, Corticotropin; D044101:Receptors, Melanocortin; D005576:Colforsin; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D002116:Calcitonin; D000242:Cyclic AMP; D008156:Luciferases; D020962:Heterotrimeric GTP-Binding Proteins; D004121:Dimethyl Sulfoxide