Applications of Tissue-specific Transcription Factor in Animal Gene Therapy

组织特异性转录因子在动物基因治疗中的应用

• 批准号: 10556071
• 负责人: INABA Mutsumi
• 金额: $ 2.24万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10556071/
• 关键词: Gene therapy; transcription factors; GATA-1; Promoters; Enhancers; Band 3; Inberited disorders; Hematopoiesis; 赤血球; 家畜

Reporter plasmid constructs were created in pSEAP vectors in which the reporter followed the transcription activating element derived from 5' upstream sequence of GATA-1 gene, including erythroid-specific promoter and exon 1, and intron 1. When transfected into K562 cells but not COS7 or CHO cells, activation of the reporter gene expression was observed. Thus, the region involving 5' upstream and intron 1 sequences was demonstrated to possess activity in enhancing gene transcription.Retroviral vector containing the 5' upstream and intron 1 sequence from GATA-1 gene fused to normal bovine band 3 cDNA (pLNCX2bebWT) was prepared and several clones of K562 cells (K562bebWT) were isolated by G418 selection. Immunofluorescence staining showed that band 3 proteins localized to the plasma membrane, whereas the mutant construct (R664X) exhibited no significant signals at the cell surface. K562bebWT showed DIDS-sensitive chloride transport, demonstrating the protein have normal function. Introduction and expression of band 3 gene into bone marrow cells from affected animals are under investigation.

在 pSEAP 载体中创建报告质粒构建体，其中报告基因跟随源自 GATA-1 基因 5' 上游序列的转录激活元件，包括红系特异性启动子和外显子 1 以及内含子 1。当转染至 K562 细胞而非 COS7 细胞时或 CHO 细胞中，观察到报告基因表达的激活。因此，涉及5'上游和内含子1序列的区域被证明具有增强基因转录的活性。制备了含有与正常牛带3 cDNA融合的GATA-1基因的5'上游和内含子1序列的逆转录病毒载体(pLNCX2bebWT)通过G418选择分离出K562细胞（K562bebWT）的几个克隆。免疫荧光染色显示带 3 蛋白定位于质膜，而突变体构建体 (R664X) 在细胞表面没有表现出明显的信号。 K562bebWT 显示 DIDS 敏感的氯离子转运，表明该蛋白具有正常功能。正在研究将带 3 基因引入受影响动物的骨髓细胞并进行表达。

项目成果

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Takahashi, S.、shimizu, R.、Suwabe, N.、Kuroha, T.、Yoh, K.、Ohta, J.、Nishimura, S.、Lim, K.-C.、Engel, J.D. 和 Yamamoto，

Nunomura, W., Takakuwa, Y., Tokimitsu, R., Krauss, S.W., Kawashima, M., and Mohandas, N.: "Regulation of CD44-protein 4.1 interaction by Ca^<2+> and calmodulin. Implications for modulation of CD44 ankyrin interaction."J.Biol.Chem.. 272. 30332-30328 (1999)

Nunomura, W.、Takakuwa, Y.、Tokimitsu, R.、Krauss, S.W.、Kawashima, M. 和 Mohandas, N.：“Ca^2 和钙调蛋白对 CD44-蛋白 4.1 相互作用的调节。调节的意义

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Inaba, M：“第 156 章：红细胞膜缺陷。Schalm 的兽医血液学，第 5 版（Feldman, R.F.、Zinkl, J.G. 和 Jain, N.C.eds.）”Lippincott Williams 和 Wilkins，纽约。

Matsumoto,M、他2名: "Molecular basis of bovine red cell protein 4.2 polymorphism in Japanese black cattle"Biochem.J.. 332. 183-187 (1998)

Matsumoto，M 等 2 人：“日本黑牛中牛红细胞蛋白 4.2 多态性的分子基础”Biochem.J.. 332. 183-187 (1998)

稲葉 睦、他21名: "赤血球(監修 三輪史朗、編集 藤井寿一、高桑雄一)" 医学書院、東京, 257 (1998)

Mutsumi Inaba 等 21 人：“红细胞（由 Shiro Miwa 监督，Juichi Fujii 和 Yuichi Takakuwa 编辑）” Igakushoin，Tokyo，257（1998）