Hatching of avian embryos under artificial environment and establishment of transgenic chicken

人工环境下禽胚胎的孵化及转基因鸡的建立

• 批准号: 09555251
• 负责人: IIJIMA Shinji
• 金额: $ 8万
• 依托单位: Graduate School of Engineering, Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09555251/
• 关键词: Quail; Chicken; Embryo; DNA transfection; Ovalbumm; Transgenic Chicken; 孵化

This year, we studied the conditions for gene transfection to embryos. For this, we applied liposome method for embryos of various developmental stages. DNA and liposome complex was injected from heat to blood vessel. Embryos after 48h fertilization gave both the highest survival and transfection efficiency. In this experiment, only heart cells showed higher expression of the introduced gene.Then we improved this point by using protamine. For this hydrophobic residues were coupled to promine and liposome protamine complex was stabilized. By using this modified liposome, the area which showed the expression of introduced DNA was expanded.On the other hand, we developed the vector system to achieve oviduct spcific gene expression. For this we cloned ovalbimin genes from chicken fibroblast DNA.From 100,000 recombinant phages, we isolated a phage containing ovalbumin promotor region. Human insulin gene DNA was ligated to the promoter region to construct the expression vector.

今年，我们研究了基因转染胚胎的条件。为此，我们将脂质体方法应用于不同发育阶段的胚胎。将DNA和脂质体复合物从热注射到血管中。受精48 h后的胚胎存活率和转染效率最高。在本实验中，只有心脏细胞表现出导入基因的高表达，然后我们用鱼精蛋白改进了这一点。为此，将疏水性残基偶联到鱼精蛋白上，并稳定脂质体鱼精蛋白复合物。利用这种修饰的脂质体，一方面扩大了导入DNA的表达区域，另一方面也为实现输卵管特异性基因的表达建立了载体系统。为此，我们从鸡成纤维细胞DNA中克隆了卵清蛋白基因，并从10万个重组质粒中分离了一个含有卵清蛋白启动子区的噬菌体。将人胰岛素基因DNA连接到启动子区，构建表达载体。

项目成果

Jun You,Masamichi Kamihira and Shinji Iijima: "Enhancement of transfection efficiency using ligand-modified lipid vesicles" Journal of Fermentation and Bioengineering. Vol.85(5). 525-528 (1998)

Jun You、Masamichi Kamihira 和 Shinji Iijima：“使用配体修饰的脂质囊泡增强转染效率”发酵与生物工程杂志。

Masamichi Kamihira, Jun You and Shinji Iijima: "Surfactant-mediated gene transfer for mammalian cells" Animal Cell Technology. 8. 381-385 (1997)

Masamichi Kamihira、Jun You 和 Shinji Iijima：“哺乳动物细胞的表面活性剂介导的基因转移”动物细胞技术。

Jun You, Masamichi Kamihira and Shinji Iijima: "Enhancement of transfection efficiency using ligand-modified lipid vesicles" Journal of Fermentation and Bioengineering. Vol.85(5). 525-528 (1998)

Jun You、Masamichi Kamihira 和 Shinji Iijima：“使用配体修饰的脂质囊泡增强转染效率”发酵与生物工程杂志。

Masamichi Kamihira,Satoshi Oguchi,Ayuko Tachibana,Yuki Kitagawa and Shinji Iijima: "Improved hatching for in vitro quail embryo culture using sarrogate eggshell and arti-ficial vessel" Development Growth and Differentiation. Vol.40. 449-455 (1998)

Masamichi Kamihira、Satoshi Oguchi、Ayuko Tachibana、Yuki Kitakawa 和 Shinji Iijima：“使用沙罗格蛋壳和人工容器改进体外鹌鹑胚胎培养的孵化”发育、生长和分化。

Masamichi Kamihira,Satoshi Oguchi,Ayuko Tachibana,Yuki Kitagawa and Shinji Iijima: "Improved hatching for in vitro quail embryo culture using sarrogate eggshell and artificial vessel" Development Growth and Differentiation. Vol.40. 449-455 (1998)

Masamichi Kamihira、Satoshi Oguchi、Ayuko Tachibana、Yuki Kitakawa 和 Shinji Iijima：“使用沙罗格蛋壳和人工容器改进体外鹌鹑胚胎培养的孵化”发育、生长和分化。