Development of systems for cell culture and expression vector to generate transgenic marine invertegra

开发细胞培养和表达载体系统以产生转基因海洋无脊椎动物

• 批准号: 08558080
• 负责人: SHIMADA Hiraku
• 金额: $ 12.67万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08558080/
• 关键词: sea urchin; pearl oyster; transgenic animal; insulator; expression vector; cell culture; 遺伝子導入; 遺伝子組換え; バフンウニ; 遺伝子; 転写調節; 転写因子

1. Identification of cis-active elements that regulate expression of sea urchin gene :Several sequence elements that regulate transcription of the sea urchin Ars gene were detected by reporter assay around its transcription start site. Among them between Sox-binding sitebetween -168bp and -162bp is most prominent element. Around -186bp cis-element that represses the promoter activity was detected.2. Validity of insulator element in gene transfer experiments :An insulator element present at the upstream of the sea urchin Ars gene showed insulator activity also in cells of Drosophila, mouse and humane3. Cell culture system for mantle epitermal cells of pearl oyster :Primary cell culture system for mantle epitermal cells of pearl oyster was established, though they do not proliferate in vitro.4. Culturing of sea urchin embryonic cells :Though we succeeded in establishing in vitro culture of cells from sea urchin pluteus larvae, we failed to culture them beyond three days because of difficulty in preventing proliferation of contaminating fungi.

1.调节海胆基因表达的顺式活性元件的鉴定：通过报告基因检测，在海胆Ars基因转录起始位点周围检测到了几个调节海胆Ars基因转录的序列元件。其中Sox结合位点之间的-168bp和-162bp之间是最突出的元件。在-186bp左右检测到抑制启动子活性的顺式元件。2．基因转移实验中绝缘子元件的有效性：海胆Ars基因上游存在的绝缘子元件在果蝇、小鼠和人的细胞中也显示出绝缘子活性。珍珠贝套膜上皮细胞培养体系：建立了珍珠贝套膜上皮细胞体外不增殖的原代细胞培养体系。 4．海胆胚胎细胞的培养：虽然我们成功地建立了海胆幼虫细胞的体外培养，但由于难以防止污染真菌的增殖，我们未能将其培养超过三天。

项目成果

Ishii et al: "Hboxl and Hbox7 are involved in the Pattern Formation of Sea urchin Embryo" Development Growth & Differentiation. 41 (印刷中). (1999)

Ishii 等人：“Hbox1 和 Hbox7 参与海胆胚胎的模式形成”发育生长和分化 41（出版中）。

Akasaka et al: "Oral-aboral ectoderm differntiation of the sea urchin embryos is disrupted in response to calcium ionophore." Development Growth and Differentiation. 39 (in press). (1997)

Akasaka 等人：“海胆胚胎的口-口外胚层分化因钙离子载体而被破坏。”

N.Sakamoto: "Two isoforms of orthodenticle-related proteins (HpOtx) bind to the enhancer element of sea urchin arylsulfatase gene." Developmental Biology. 181. 284-295 (1997)

N.Sakamoto：“正牙齿相关蛋白 (HpOtx) 的两种亚型与海胆芳基硫酸酯酶基因的增强子元件结合。”

Kawasaki,et al: "Lim1-related homeobox gene(HpLim1) expressed in sea urchin embryo" Development Growth&Differentiation. 41印刷中. (1999)

Kawasaki 等人：“海胆胚胎中表达的 Lim1 相关同源盒基因 (HpLim1)”，出版中的《发育生长与分化》(Development Growth&Differentiation) 41。

Mitsunaga-Nakatsubo et al: "Arylsulfatase exists as non-enzymatic cell surface protein in sea urchin embryos" Journal of Expermintal Zoology. 280. 220-230 (1998)

Mitsunaga-Nakatsubo 等人：“芳基硫酸酯酶作为非酶促细胞表面蛋白存在于海胆胚胎中”《实验动物学杂志》。