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转基因鱼的定点整合研究
汪亚平
Subtype博士
Thesis Advisor朱作言
2000
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Keyword转植基因 定点整合 荧光筛选 随机整合 遗传分析 快速生长 基因组靶位
Abstract转植基因(transgene)在受体鱼基因组中以多拷贝串联方式的多位点随机整合,严重制约转基因鱼纯系的快速建立和转植基因的遗传稳定性,是转基因鱼研究亟待解决的问题。转植基因定点整合技术是解决上述问题的重要途径。转植基因定点整合技术,即基因打靶技术,在小鼠基因功能研究中得到广泛应用,并取得重要研究进展。本研究旨在针对鱼类胚胎发育特点,建立鱼类转植基因定点整合技术,以实现转植基因在受体鱼基因组有效靶位的单拷贝定点整合,籍此快速培育转植基因稳定整合的转基因鱼纯系。鱼类植基因定点整合研究包括两个方面,其一是建立定点整合技术,实现转植基因在基因组既定靶位的高效整合;其二是克隆鱼类基因组有效整合位点,为转植基因的定点整合提供基因组靶位。本文就此两方面进行了探索,主要研究内容如下:1.基于鲤鱼β-actin基因顺序,采用长片段PCR技术,克隆了海鲤、大黄鱼的β-actin基因DNA片段,以此作为基因组定点整合靶位。2.根据靶位定点整合正负筛选(PNS)和正向筛选(PS)原理,以EGFP基因为筛选基因,采用启动子缺失方案,设计构建了荧光正向筛选靶位定点整合载体(FPS)。同时构建了药物正负筛选靶位定点整合载体。3.采用组织培养方法,获得鲤鱼和海鲤的尾鳍原代培养细胞,以GFP基因为报道基因,进行了培养细胞DNA电脉冲转染条件优化。4.在鲤鱼培养细胞中进行了转植基因的定点整合研究,采用荧光正向筛选方法,检测到转植基因定点整合的绿色荧光细胞,其出现频率约为10~(-5);采用药物正负筛选方法,获得抗性细胞克隆,其出现频率为10~(-5)-10~(-6),PCR检测证实,转植基因在基因组靶位实现了定点整合。5.在鲤鱼转基因胚胎中进行了转植基因的定点整合研究,采用荧光正向筛选方法,在转基因胚胎肌肉效应期,部分胚胎中检测到绿色荧光细胞团,绿色荧光细胞嵌合体胚胎的出现频率约为1%(28/2653),PCR检测证实,转植基因在基因组靶位实现了定点整合,PCR检出率约为1‰(2/2653)。6.采用显微注射方法,研制转“全鱼”GH基因黄河鲤鱼,获得快速生长个体。通过与对照黄河鲤鱼杂交,从子代中筛选出快速生长的个体,并对杂交子代转植基因分离进行了遗传分析。结果表明,外源基因在转基因鱼P_o代2-3条染色体上整合,不同整合位点转植基因促生长效应存在差异。本研究首次建立了鱼类转植基因定点整合技术,针对鱼类胚胎发育特点,采用荧光筛选方法,进行鱼类转基因胚胎的转植基因定点整合研究,验证了鱼类转植基因定点整合研究的新思路。在转“全鱼”GH基因鱼的转植基因整合位点研究中,首次应用遗传分析方法,证实转植基因在受体鱼中的多位点整合;通过杂交子代生长数据的统计分析,证实转植基因生物学效应的多样性,同时在转植基因分离的杂交子代中筛选出快速生长个体,为进一步筛选有效整合转植基因,以及基因组有效靶位顺序的克隆奠定了基础。
Other AbstractTransgenes are normally integrated randomly in multi-copies at various sites of host fish genome, so that the establishment of homogenous strains of transgenic fish and the genetic stability of transgenes are highly influenced. This has been one of the most important problems to be solved in transgenic fish. The site-specific integration of transgenes, known as gene targeting, is a reasonable and important method to deal with this problem. The art of gene targeting has been exclusively studied and extensively applied in the study of gene functions in mouse, and many important results have been achieved. Our goal is to establish the gene-targeting technique in fish in consideration of the characteristics of fish embryogenesis. Thereby, the homogenous strain of transgenic fish may be quickly produced along with the success of site-specific integration of a single copy transgene at the target site in fish genome. The study of gene targeting in fish includes two major parts. First, transgenes can be effectively integrated into the specific site of host genome by the achievement of techniques in gene targeting. Second, the target site for the site-specific integration in fish genome may be obtained through the cloning of DNA sequences of effective integration sites. These have therefore been investigated in the present study, with the major results indicated as follows. 1. Based on the reported DNA sequence of common carp (Cyprinus carpio) β-actin gene, long-length PCR was employed to clone the β-actin gene DNA sequences of Cyprinus acutidorsalis and Pseudosciaena crocea as the target sites for gene targeting. 2. According to the principles of positive and negative selections (PNS) and also the positive selection (PS) for gene targeting, the gene targeting vectors with a strategy of florescent positive selection (FPS) were designed and subsequently constructed, with EGFP as a selective marker and promoter-capture as a selective approach. Meanwhile, a PNS vector for drug selection was also constructed. 3. The primary cultures of tail-fin cells of common carp and C. acutidorsalis were successfully performed, and optimized parameters for electro-transformation were obtained by using GFP as a reporter in cultured cells. 4. Gene targeting was extensively studied in cultured cells of common carp. When FPS was utilized for gene targeting, green florescent cells with site-specific integrated transgene were distinctly detected (the detection rate is about 10~(-5)). On the other hand, when drug PNS was utilized, anti-drug cell clones were obtained with an efficiency of 10~(-5)-10~(-6). PCR was subsequently performed to confirm the site-specific integration. 5. As another valid approach, gene transfer in common carp embryos with FPS vectors was performed for gene targeting. A few embryos at muscular reaction stages were found to have cell masses showing green florescence, with a detection rate of about 1% (28/2655). The PCR amplification also confirmed the occurrence of site-specific integrations with a detection rate of about 11‰ (2/2655). 6. "All-fish" GH gene were microinjected into the fertilized eggs of Yellow River carp (Cyprinus carpio), and "fast-growing" transgenic fish were successfully produced. By cross-breeding with non-transgenic controls, the F1 generation with the transgene was obtained, and the "fast-growing" individuals were then selected. The results of the genetic analysis on the genetic separation of transgenes in the F1 generation revealed that transgenes were randomly integrated into the host genome at 2 to 3 chromosomes. Statistical analyses on the growth in transgenic fish and non-transgenic siblings confirmed that GH-transgene can significantly stimulate fish growth, while different-site-integrated transgenes have distinct biological effects. In conclusion, techniques for gene targeting in fish have been established in this pilot study. Considering the characteristics of fist embryogenesis, the application of florescent selection has, for the first time, been employed in the site-specific integration research on FPS vector-transferred fish embryos. This provides a new area for the site-specific integration of transgenes in fish. In the study of integration sites of "all-fish" GH gene transgenic fish, genetic analyses were firstly employed to prove the multi-site integrations of transgenes in host fish. Using statistical analyses on growth parameters of the hybrid offspring, the polymorphism of biological effects was confirmed. Meanwhile, "fast-growing" individuals among the F1 generation were selected, which provides a steady basis for the further cloning of DNA sequences of functional integrated transgenes and effective target sites.
Pages105
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12566
Collection学位论文
Recommended Citation
GB/T 7714
汪亚平. 转基因鱼的定点整合研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2000.
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