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转基因克隆牛外源基因整合位点及拷贝数研究

标题: 转基因克隆牛外源基因整合位点及拷贝数研究
英文标题: Transgene Integration Site and Copy Number Analysis in Ttansgenic Cloned Bovine
作者: 谭贝贝
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 转基因克隆牛,TAIL-PCR,DWACP-PCR,整合位点,绝对定量PCR,拷贝数,纯合性
英文关键词: transgenic cloned bovine,TAIL-PCR,DWACP-PCR,integration site,absolute quantitative PCR,copy number,zygosity
论文级别: 硕士
学位: 学位论文
导师: 李世杰
专业: 微生物与生化药学
提交时间: 2012
摘要: 转基因克隆动物是在获得转基因细胞系的基础上进行体细胞克隆(体细胞核移植),生产具有特定性状、特殊功能或者一定目的的克隆动物群.近十年来,转基因技术与体细胞克隆技术的结合使转基因动物的生产有了突飞猛进的发展.转基因克隆动物在生产人类重要的医药蛋白、培育抗病品系、异种器官移植以及基因治疗等方面都显示出了广阔的应用前景.确定外源基因的整合位点及拷贝数是对外源基因下一步表型研究和整合机理探讨的前提条件.本实验首先分析了转人溶菌酶基因克隆牛中外源基因的整合位点及拷贝数,并分析了转基因个体整合位点的纯合性.本实验得到结果如下:1.应用TAIL-PCR、DWACP-PCR和旁侧PCR方法得到了2头转基因克隆牛外源基因的整合位点,结果表明2个转基因个体中外源基因均以单一位点形式整合在受体基因组上,得到两个整合位点即4个结合片段.2.0504个体外源基因整合在牛的24号染色体的基因组克隆(NW003104566.1)中.外源基因整合造成染色体约238bp的核苷酸缺失,外源基因3′端有约3103bp核苷酸的缺失且整合位点处富含AT序列.2个整合片段的末端与拓扑异构酶I作用位点的共有序列相连.外源基因与染色体的整合连接处存在短的同源序列.3.Yun个体中外源基因整合在牛的16号染色体的基因组克隆(NW003104440.1)中,整合位点位于LOC10030基因和RGL1基因之间.外源基因的整合造成染色体约228bp的核苷酸缺失,外源基因3′端同样缺失约3103bp的核苷酸.整合位点连接处同样导致个别碱基的缺失.2个整合片段的末端与拓扑异构酶I作用位点的共有序列相连.并且外源基因与染色体的整合连接处存在短的同源序列.4.本实验以GAPDH基因为内参,采用绝对定量PCR法检测外源基因拷贝数.标准曲线为:Log2N (拷贝数) = -0. 3075△Ct+0.7421 (R2=0.9996, p<0.001),计算得到4头原代转基因克隆牛外源基因拷贝数分别为1.335883、1.234408、1.169825、1.0127,说明外源基因基本以单拷贝形式整合到受体基因组上.5.采用整合位点5′上游和3′下游侧翼序列特异性引物与外源基因特异性引物的组合,进行旁侧PCR,分析整合位点的纯合性.检测到转基因克隆牛与野生型牛一致的侧翼序列特异性引物扩增片段,表明2头转基因克隆牛均为整合位点杂合子.
英文摘要: Transgenic and cloned animals are defined to clone animals on the basis of transgenic animals, and to produce cloning fauna with specific traits, special function or certain purpose. It is the organic combine of transgenic animals and cloned animals.Recent progress in animal cloning has provided an attractive alternative to improve transgenic efficiency, through the combination of transfection and somatic cell nuclear transfer (SCNT). Transgenic animals have been used to produce important proteins, livestock bioreactor, study copy number, integration site, cultivate the disease-resistant strains, xenotransplantation donor and production of bio-pharmaceutical products. It has shown a broad application prospects.After establishment of transgenic animal line, it is important to detectand integration site and transgene copy number, and it is necessary to determine transgene expression and function. Therefore, we checked transgene integration site and copy number in transgenic bovine. Moreover, Junction PCR was employed to confirm the integration site and analyze zygosity.The results were as follows:1. Integration site was successfully cloned by TAIL-PCR, DWACP-PCR and Junction PCR. The results showed that integration of exogenous genes to bovine chromosome in single one point integration. For integration sites were detected by BLAST.2.The results showed that integration of 0504 exogenous genes to bovine chromosome 24 genomic clones (NW003104566.1). Exogenous gene integration caused about 238bp nucleotide in chromosome missing, about 3103bp nucleotide in exogenous gene 3′end missing and AT-rich sequence in intergration site. And two junctions were associated with the consensus sequence for topoisomerase-I cleavage sites. At two genome-transgene junctions, short homologies were found.3. The results showed that integration of Yun exogenous genes to bovine chromosome 16 genomic clones (NW003104440.1), between gene LOC10030 and gene RGL1.Exogenous gene integration caused about 228bp nucleotide in chromosome missing, about 3103bp nucleotide in exogenous gene 3′end missing. Analysis of two genome-transgene junctions showed that'nibbling'of ends had occurred at some ends to joining. And two junctions were associated with the consensus sequence for topoisomerase-I cleavage sites. At two genome-transgene junctions, short homologies of 1 nt were found.4. Absolute quantitative PCR was used to calculate transgene copy number. The parameters of the standard curve were: Log2N=-0.3075△Ct+0.7421 (R2=0.9996, p<0.001), and copy numbers were 1.335883, 1.234408, 1.169825, 1.0127. Exogenous gene basic as a single copy integrated into the genome.5. Junction PCR combining with 5′and 3′integration site and transgene specific primers was performed to analyze integration site zygosity. Bands amplified by 5′and 3′integration site specific primers just as WT control were obtained to determine the heterozygosity of integration site.