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

今年，我们研究了胚胎基因转染的条件。为此，我们将脂质体法应用于不同发育阶段的胚胎。将DNA和脂质体复合物热注入血管。受精48h后的胚胎存活率和转染效率最高。在这个实验中，只有心脏细胞表现出较高的基因表达。然后我们用鱼精蛋白改善了这一点。疏水残基与脯氨酸和脂质体偶联，稳定了鱼精蛋白络合物。利用该修饰脂质体，扩大了导入DNA的表达区。另一方面，我们开发了载体系统来实现输卵管特异性基因的表达。为此，我们从鸡成纤维细胞DNA中克隆了卵白蛋白基因。从10万个重组噬菌体中分离出一个含有卵清蛋白启动子区的噬菌体。将人胰岛素基因DNA连接到启动子区域构建表达载体。

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.