Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术，可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现，体外重叠PCR法难以获得高质量的目的DNA打靶片段，严重影响重组效率。为了解决上述问题，根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段，然后将其电击共转化进入携带Red重组酶和pcDNA3．1载体DNA的大肠杆菌DY331菌株内，经体内同源重组直接产生的pcDNA3.1—egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得，阳性率可达到45％。瞬时转染pcDNA3．1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。

Gene Fusion of egfp & kan and Recombinant Plasmid Construction by Red Mediated in vivo Homologous Recombination

Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR, therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.