利用多位点基因打靶技术进行鳜鱼转干扰素基因的研究

The project is a small project of National Nature Science Foundation for 1 year. It was planed to get the interval sequence of rDNA genes of siniperca chuatsi and to construct the gene targeting vector for multiple site. Some rDNA sequences of oreochromis esculentus and tetraodon nigroviridis have been got by the.bioinformatics techniques. The contig were constructed with these sequences. Two.primers were designed in the conserved regions of the contig. A 3.1kb sequence of.rDNA and its interval sequence of siniperca chuatsi has been amplified and cloned by LA Taq polymerase with GC buffer. Two interval sequences of ITS1 and ITS2 have been got and confirmed by sequencing. The 2.7kb HSV-TK gene were inserted at the end of 3.1kb. The gene targeting vector of multiple site for siniperca chuatsi have been constructed. The key material of gene targeting technique of multiple site for.siniperca chuatsi has been got in the project. The project has got the further financial aid from Science and Technology Developing Foundation of Foshan overnment.

利用申请者建立的多位点基因打靶技术，以鳜鱼细胞中多拷贝rDNA基因的间隔序列作为引导序列和靶位点，构建基因打靶载体进行转基因，获得多位点定点整合干扰素基因的鳜鱼。建立一套适用于鱼类等动物进行基因定点整合的外源基因随机插入所致目的基因不能稳定遗传合表达、毒性整合、整合效率低等问题。