| Summary: | 碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 96 === This study is trying to set up an estrogen receptor-mediated GFP reporter gene system which can be used to detect estrogenic compounds abounded in environment. First of all, estrogen responsive elements (EREs) fragments were amplified with TR-PCR method and inserted into pGEM-T Easy vector. These resulting products, including pGEM-3ERE, pGEM-4ERE, pGEM-6.5ERE, and pGEM-7ERE, were named by their total numbers of EREs. Second, pGEM-3, 4, 6.5, 7 ERE were digested with EcoR I/BamH I, and then 3, 4, 6.5, and 7 ERE-copies products were cloned into pEGFP-1 vector. Third, thymidine kinase (TK) promoter fragments were inserted into pEGFP-3, 4, 6.5, 7ERE plasmids. Forth, the expression plasmids pEGFP-3, 4, 6.5, 7ERETK were transfected into human breast cancer cell line, MCF-7, and these final cell lines were named MCF-3ERETK, MCF-4ERETK, MCF-6.5ERETK, and MCF-7ERETK. Finally, estrodial (estrogen agonist) and ICI (estrogen antagonist) were introduced to validate these assay systems. As a result, we demonstrate that multiple EREs can increase transcriptional activity. In addition, well-known estrogenic chemicals, BPA and NP, were also introduced and resulted in significant EGFP expression ranging from 0.01 to 10 μM in MCF-3ERETK and MCF-7ERETK cell lines. In the future, MCF-3, 4, 6.5, 7 ERETK cell lines could be used for screening potential environmental estrogenic compounds around environment.
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