title: The Transgenic Zebrafish Display Fluorescence Reflecting the Expressional Dynamics of Dihydrofolate Reductase.
abstract: Dihydrofolate reductase (DHFR) reduces folic acid and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is upregulated in rapidly proliferating cells and hence a favored target of antifolate drug against cancers, autoimmune diseases, and microbial infections. However, increased expression of dhfr contributed to the often emerging drug resistance and impeded the therapeutic efficacy of antifolate drugs. Therefore, comprehensive knowledge on the expressional control of dhfr becomes crucial. We generated two zebrafish transgenic lines, Tg(zdhfr+91:EGFP) and Tg(zdhfr+79:EGFP), which express green fluorescent protein driven by two zebrafish dhfr promoter fragments separately. The fluorescence intensity displayed in these transgenic embryos recapitulated the expressional dynamics of endogenous dhfr and reflected changes in dhfr mRNA and protein levels. The fluorescence intensity of these transgenic embryos was responsive to both genetic and environmental factors potentially modulating dhfr promoter activity. Sequence analyses revealed partial conservation on the landscape of transcription factor arrangement between zebrafish and human dhfr promoters. A noncanonical and inhibitory Sp1 site was identified 170 base-pair upstream to the conserved Sp1 site in close proximity to the translation initiation codon. Our results supported the potential use of these transgenic embryos for studying the expressional dynamics of dhfr and preliminary screening for dhfr promoter modulators.
authors.affiliation: 1 The Institute of Basic Medical Science, National Cheng Kung University , Tainan, Taiwan .
authors.forename: Wen-Ni
authors.initials: WN
authors.lastname: Chang
authors.affiliation: 1 The Institute of Basic Medical Science, National Cheng Kung University , Tainan, Taiwan .
authors.forename: Wan-Yu
authors.initials: WY
authors.lastname: Chi
authors.affiliation: 1 The Institute of Basic Medical Science, National Cheng Kung University , Tainan, Taiwan .
authors.forename: Tseng-Ting
authors.initials: TT
authors.lastname: Kao
authors.affiliation: 2 Department of Medical Laboratory and Biotechnology, Chung Shan Medical University , Taichung, Taiwan .|3 Department of Clinical Laboratory, Chung Shan Medical University Hospital , Taichung, Taiwan .
authors.forename: Jen-Ning
authors.initials: JN
authors.lastname: Tsai
authors.affiliation: 4 Department of Biotechnology, Kaohsiung Medical University , Kaohsiung, Taiwan .
authors.forename: Wangta
authors.initials: W
authors.lastname: Liu
authors.affiliation: 4 Department of Biotechnology, Kaohsiung Medical University , Kaohsiung, Taiwan .|5 The Institute of Biomedical Sciences, National Sun Yat-sen University , Kaohsiung, Taiwan .
authors.forename: Shih-Shin
authors.initials: SS
authors.lastname: Liang
authors.affiliation: 4 Department of Biotechnology, Kaohsiung Medical University , Kaohsiung, Taiwan .|6 Department of of Biological Sciences, National Sun Yat-sen University , Kaohsiung, Taiwan .
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Chiu
authors.affiliation: 4 Department of Biotechnology, Kaohsiung Medical University , Kaohsiung, Taiwan .|5 The Institute of Biomedical Sciences, National Sun Yat-sen University , Kaohsiung, Taiwan .|7 Department of Medical Research, Kaohsiung Medical University Hospital , Kaohsiung, Taiwan .
authors.forename: Bing-Hung
authors.initials: BH
authors.lastname: Chen
authors.affiliation: 1 The Institute of Basic Medical Science, National Cheng Kung University , Tainan, Taiwan .|8 The Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University , Tainan, Taiwan .
authors.forename: Tzu-Fun
authors.initials: TF
authors.lastname: Fu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D005453:Fluorescence; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D016329:Sp1 Transcription Factor; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase
keywords: dihydrofolate reductase; folate; one-carbon metabolism; promoter; transcriptional regulation; zebrafish