Isolation of estrogen-responsive genes of human osteosarcoma MG-63 cells

Objective To obtain a series of differentially expressed cDNA fragments from osteosarcoma MG 63 cells induced by 17 beta estradiol and to find some estrogen responsive genes. Methods Optimized cDNA representational difference analysis (RDA) was performed to isolate up regulated expressed sequences between cDNAs from MG 63 cells treated with and without 17 beta estradiol. The sources of up regulated expressed cDNA fragments were proved by Southern blot and shotgun Northern blot. The fragments were cloned into the pGEM T easy vector. After blue white selection, white colonies were picked up and individually grown in LB/Amp + medium. Some white colonies were randomly selected for DNA sequencing, homology analysis,and Northern blot analysis. Results Five up regulated expressed fragments were isolated in the 4th subtraction hybridization by cDNA RDA. These fragments proved to be really up regulated expressed and really came from the cDNAs from 17 beta estradiol treated MG 63 cells by Southern blot and shotgun Northern blot. Twenty five clones were sequenced and 13 sequences were obtained. Seven sequences were highly homologous to known genes and 2 of them proved to be differentially expressed through Northern blot. Conclusion cDNA RDA is one of the most effective methods to isolate differentially expressed genes. Some genes expression of human osteosarcoma MG 63 cells are up regulated and induced by 17 beta estradiol, and it may be related to the pathogenesis of postmenopausal osteoporosis.