[Isolation of differentially expressed genes in human osteoblast-like osteosarcoma cell line induced with...

To obtain a serial differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced with 17beta-estradiol and to find some estrogen-responsive genes.

Optimized cDNA representational difference analysis (RDA) was performed to isolate up-regulated expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of up-regulated expressed cDNA fragments were proved by Southern blot. The fragments were cloned into the pGEM-T easy vector and a cDNA library was prepared in E. coli JM109 cells. The cDNA library was plated on LB/Amp(+)/X-gal/IPTG plates and white colonies were picked up and individually grown in LB/Amp(+) medium in 96-well plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with alpha-(32)P-labeled subtracted or unsubtracted tester cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones were selected for DNA sequencing, BLAST and Northern blot analysis after release of the pGEM-T easy insert with EcoR I.

Five up-regulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced with 17beta-estradiol as tester amplicon and cDNA from untreated MG-63 cells as driver amplicon by cDNA RDA. These fragments were proved to be really coming from tester amplicon and down-regulated expressed in the untreated cell by Southern blotting. We obtained more than 600 cDNA clones with positive insert from MG-63 cells line induced with 17beta-estradiol and about 120 differentially expressed clones through dot blotting. Twenty clones were sequenced and we got 15 gene sequences. One of them was proved to be differentially expressed through Northern blotting.

cDNA RDA is one of the most effective methods which can isolate differentially expressed genes and we can screen differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some genes of human osteoblast-like osteosarcoma MG-63 cell line showed up-regulated expression after induction by 17beta-estradiol.