講演情報
[II-JSCDJS-P1-5]レポーターゼブラフィッシュを用いた、EP4発現調節機序の解明
○吉田 賢司1,2, 内田 敬子1, 南沢 享1,3, 川原 玄理4, 林 由起子4, 横山 詩子1 (1.東京医科大学 細胞生理学分野, 2.東京慈恵会医科大学 小児科学講座, 3.ビューティ＆ウェルネス専門職大学, 4.東京医科大学 病態生理学分野)
キーワード：
EP4、reporter zebrafish、drug screening
Background: Prostaglandin E2 receptor, EP4, expression increases in the ductus arteriosus (DA) at birth and greatly contributes to its closure. However, the molecular mechanisms regulating EP4 expression remain unclear. Zebrafish lay large number of eggs, and transparent larvae allow high-throughput screening and direct observation of the developmental process with fluorescent reporters.
Objective: The aim of this study was to establish a reporter zebrafish line to identify factors regulating EP4 expression.
Methods: We injected the upstream region of ptger4b and GFP cDNA into fertilized eggs with the Tol2 transposon system. We examined fluorescent signals of the zebrafish lines in comparison with the endogenous ptger4b expression pattern which was confirmed by in situ hybridization, and conducted drug screening using the line.
Results: Focusing on the potential promoter regions of the zebrafish ptger4b, highly homologous to the human PTGER4 gene encoding EP4, we established two transgenic lines, Lines 1246 and 315. We detected endogenous ptger4b expression in the neural crest. It was consistent with the EGFP signal in Line 1246. The drug screening using pooled compounds identified 76 candidates enhancing fluorescence intensity out of 3090 compounds in the FDA-approved drug library. Most of the drugs had purposes other than cardiovascular disease, such as infectious diseases.
Conclusion: We identified ptger4b gene expression in the neural crest, the origin of the DA, in Line 1246. We will further narrow down the candidates to identify compounds that regulate EP4 expression.
Objective: The aim of this study was to establish a reporter zebrafish line to identify factors regulating EP4 expression.
Methods: We injected the upstream region of ptger4b and GFP cDNA into fertilized eggs with the Tol2 transposon system. We examined fluorescent signals of the zebrafish lines in comparison with the endogenous ptger4b expression pattern which was confirmed by in situ hybridization, and conducted drug screening using the line.
Results: Focusing on the potential promoter regions of the zebrafish ptger4b, highly homologous to the human PTGER4 gene encoding EP4, we established two transgenic lines, Lines 1246 and 315. We detected endogenous ptger4b expression in the neural crest. It was consistent with the EGFP signal in Line 1246. The drug screening using pooled compounds identified 76 candidates enhancing fluorescence intensity out of 3090 compounds in the FDA-approved drug library. Most of the drugs had purposes other than cardiovascular disease, such as infectious diseases.
Conclusion: We identified ptger4b gene expression in the neural crest, the origin of the DA, in Line 1246. We will further narrow down the candidates to identify compounds that regulate EP4 expression.