作　　者： ; ; ; ; ; ; ; ; ;

机构地区： 暨南大学

出　　处： 《中国水产科学》 2005年第4期377-382,共6页

摘　　要： 以鳜鱼(Sinipercachuatsi)为研究对象,以其重复的rRNA基因间的间隔序列为靶位点构建多位点基因打靶载体,为建立体内多位点基因打靶技术获得关键材料。先构建含TK和Neo基因的通用打靶载体pCTKNeo。采用LATaq高保真酶扩增获得左、右同源重组引导臂HRDS1、HRDS2,经T质粒克隆后,分别插入到pCTKNeo载体Neo基因的上下游,构建成鳜鱼专用的多位点打靶载体pCTKNeoHRDS1/2。同样将干扰素基因hIFN插入到pCTKNeoHRDS1/2载体的Neo基因与HDRS2之间。每次克隆均经PCR、酶切和测序等鉴定DNA片段的插入及插入方向,最终构建成多位点基因打靶载体pCTKNeoHRDS1/2hIFN。目前的基因打靶技术存在打靶效率低、安全性等问题,以重复序列为靶位点的多位点基因打靶技术将部分解决这些问题。 The target loci of gene targeting are usually single copy sequences. The DNA repetitive sequences in genome are thought as forbidden zone of gene integrating. The authors have set up the techniques of multiple locus gene targeting using repetitive sequences as target loci in vitro. The no-coding repetitive sequences in NOR(nucleolus organization region) of animal cell were used as target loci. There were some problemes such as low efficiency and safe problem in the gene targeting. The technique of multiple locus gene targeting on repetitive sequences would resolve part of these problems. Siniperca chuasti was used as the object in this research. The vectors of multiple locus gene targeting on repetitive internal transcribed spacers between rRNA genes were constructed. The key material of multiple locus gene targeting in vivo would be got.The general vector of gene targeting of pCTKNeo with TK gene and Neo gene was constructed firstly. The SP1416-T plasmid of rRNA genome of Siniperca chuatsi was got by cloning and PCR amplification with LA Taq polymerase. Two pairs of primers of HRDS1U/HRDS1D and HRDS2U/HRDS2D were designed. The two arms of HRDS1(homogenous recombination direct sequence) and HRDS2 of the targeting vector were amplified by PCR using the SP1416-T plasmid as the templet. The PCR products of HDRS1 and HDRS2 were cloned to T-vector. The HRDS1 and HRDS2 digested from the HRDS1-T and HRDS2-T by Nhe Ⅰ/Mfl Ⅰ and BamH Ⅰ/Bgl Ⅱ were inserted into the upstream and downstream of the Neo gene of pCTKNeo vector respectively. The pCTKNeo-HRDS1/2 vector of multiple locus gene targeting for Siniperca chuatsi was constructed. The other pair of primers of IF1/IF2 were also designed. The human interferon gene (hIFN) was amplified by PCR using the pCDNA3-hIFN plasimid as the templet. The PCR products of hIFN gene were cloned into T-vector. The hIFN gene digested from the hIFN-T by BamH Ⅰ/Bgl Ⅱ was inserted into the BamH Ⅰ site between Neo gene and HRDS2 of pCTKNeo-HRDS1/2. The insertion and the insert dire

关 键 词： 基因打靶 多基因座 载体 重复序列 鳜鱼

领　　域： [生物学]