SELECTION OF GLUCOCORTICOID-RESISTANT MUTATIONS FROM AN ATT-20 CELL-LINE CONTAINING A GLUCOCORTICOID-REGULATED SELECTABLE TRANSGENE

AtT-20/IDG8 cells contain the stably transfected, selectable gene, neo mycin phosphotransferase, under negative glucocorticoid regulation. Th us, when cultured in the simultaneous presence of the neomycin analogu e, G418, and dexamethasone, AtT-20/IDG8 cells fail to grow. Our hypoth esis was that mutated AtT-20/IDG8 cells capable of growth in such medi um would have a defect in the glucocorticoid-mediated regulation of th e neo(r) gene. AtT-20/IDG8 cells were chemically mutagenized using eth yl-methane sulfonate and cloned in the presence of G418 and dexamethas one. Fourteen clones were obtained and loss of glucocorticoid control of neo(r) expression was confirmed in them all. The naturally occurrin g gene, pro-opiomelanocortin, which is down-regulated by glucocorticoi ds in parent AtT-20/1DG8 cells, was down-regulated by dexamethasone in ten of the mutant lines, indicating that in those cells the receptor was functional in spite of aberrant regulation of neo(r). In the other four lines, pro-opiomelanocortin regulation was lost,also suggesting that a general transcription factor, such as the receptor, had been al tered. These results indicate that multiple factors are involved in gl ucocorticoid-mediated gene regulation and that new, informative mutati ons can be produced after insertion of a regulated, selectable gene in to a previously non-selectable cell line. (C) 1995 Academic Press, Inc .