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题名: 转植基因鱼的核移植研究
作者: 赵浩斌
答辩日期: 1999
导师: 朱作言
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 细胞 ; 转植基因 ; 整合 ; 表达 ; 核移植 ; 同质化
摘要: 转植基因鱼研究正在从理论走向应用,转“全鱼”生长激素基因的鱼已经开始中试,大面积的推广应用己为时不远。然而,转植基因的行为的可控性和遗传的不稳定性及转植基因同质化建立等问题尚待深入研究和解决。核移植技术不失为解决这些问题的一个重要手段。本研究用核移植方法,就同质化转植基因鱼的途径及方式进行了较为系统的研究,全文共分五章。第一章:综述了近年在鱼类基因转移研究上取得的一些进展,包括核移植和胚胎二F细胞研究等。第二章:主要以泥鳅(Misgurnus anguillicaudatus Cantor)为试验鱼,以绿色荧光蛋白(GFP)基因为报告基因,研究了基因整合与表达的时序。结果表明外源基因在鱼类胚胎发育过程中的整合从囊胚期开始,基因表达的起始时间及强度与启动子有关,环形质粒的表达率高于线形质粒。结构上耳无关系的质粒共转移时,两者的整合表达之间没有直接关系。第三章:以澎泽鲫(Carassius auratus L.,Pengze var.)和黄河鲤(Cyprinus carpio, Huanghe var.)为试验对象,进行了早期转植基因胚胎细胞的核移植,得到了核移植澎泽鲫和黄河鲤。外源基因检测结果显示尚未获得克隆的转植基因鱼,其原因可能在于早期胚胎细胞中整合的只占极少一部分,因而以早期转植基因胚胎细胞核移植要获得同质化转植基因鱼十分困难。第四章:以F_4代转hGH基因红鲤体细胞(肾脏和尾鳍)及培养18代次的转hGH基因红鲤F_4代尾鳍细胞为核供体,泥鳅或黄河鲤成熟卵为受体,进行了核移植。结果表明F_4代转植基因红鲤外源基因阳性率100%,各组织均含有外源基因,F_4代转植基因红鲤中的外源hGH基因已基本稳定。F_4代红鲤肾脏细胞核与泥鳅卵配合的核移植胚胎发育到神经胚,F_4代尾鳍细胞核移入泥鳅卵后的重组胚发育到肌肉效应期。尾鳍培养细胞与黄河鲤卵子配合的重组胚胎发育到肌节期。核移植胚胎中hGH基因100%存在,说明体细胞核移植有望作为获得同质化转植基因鱼的途径。第五章:对培养的鲫鱼囊胚细胞株CAB进行了脂质体介导的ccMTeGFP基因转移,得到了抗G418的GFP基因阳性细胞,细胞形态及细胞正常的生长未受到外源基因的影响。利用基因转化的CAB细胞为供体进行了核移植,获得转植外源基因的核移植胚胎。4℃处理的细胞核移植后胚胎发育率显著增高。说明通过培养细胞的基因转化和核移植有望获得稳定整合的转植基因鱼,供体细胞的状态对核移植效率有显著影响。总之,通过上述转植基因鱼核移植的研究,探讨了获得同质转植基因鱼的途径,即通过体细胞转化、筛选和核移植的途径。有待进一步研究的问题是:①培养细胞的早期转化和筛选,以保持细胞染色体组成的正常;②胚胎干细胞的建立;③供体细胞与受体卵在细胞周期上的协调,以提高核移植的效率。
英文摘要: Transgenic fish research is in the direction of application, and the "all fish" growth hormone gene transfer fish is under conditional field trial. However, some problems remain to be solved such as uncontrollable behavior and unstable heredity oftransgene, etc. Nuclear transplantation is a major technique to solve these problems, so a study is conducted to know how to make homogenous transgenic fish by nuclear transplantion This paper includes five pats as follows. Part 1: It is a review abort achievements oftransgenic fish, nuclear transplantation and embryonic stem cell research in recent years. Part 2: Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) was studied. The GFP gene expression was first observed at the gastmla stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration ofthe GFP gene was first detected at the blastula stage and lasted for quite a long period. When two types of different plasmid were co-injected into fertilized eggs, the behavior of their integration and expression was not identical. Part 3: Nuclear transplantation was carded through with transgenic fish embryos of Carassius auratus, Pengze var and Cyprimus carpio, Huanghe var at blastula and gastrula stage as donors. Foreign gene was detected by PCR in five nuclear transplanted fish of Carassius auratu. Pengze var and one nuclear transplanted fish of Cyprinus carpio. Huanghe var. There was not any homozygote of transgenic fish acquired The limitation was analyzed about acquiring homozygote of transgenic fish by nuclear transplantation using transgenic embryos at early stage as donors, and the future strategy was put forward. Part 4: Nuclear transplantation was carded out with somatic cells including kidney cells, tail fin cells and cultured tai fin cells of F_4 hGHgene tmnsfened red carp as donors, and unfertilized eggs of loach or Huanghe carp as recipients. The results show that hGH gene stablely exists in the F_4 red carp. The recombined embryos could develop, but no one develope to term. The hGHgene exists in all nuclear transplant embryos detected by PCR. The results suggested that nuclear transfer of somatic cells might acquire cloned transgenic fish. Part 5: The ccMTeGFP plasmid was used to transfect CAB cells fiom crucian cap blastula by lipofection method, and the GFP gene positive cells were acquired by G418 selection, the cell morphology and growth were not affected by GFP gene. The pCMVeGFP or pMhGHtransfected CAB ceU nuclei were transferred into silver carp unfertilized eggs, and nuclear transfer embryos with foreign gene were acquired. The developmental ratio of nuclear transfer embryos was higher from cells incubated at 4 ℃ for 24h than from fresh cells. It is suggested that homogenous transgenic fish may be acquired through nuclear transplantation using transfected cells, and the status of donor ceils significantly affect the efficiency of nuclear transplantation. It is proposed to make homogenous transgenic fish by tranfecting cultured cells, selecting transfected cells, transferring selected cell nuclei into oocytes, and culturing nuclear transfened fishes. However, There ate some problems still retrain such as transfecting and selecting cells earlier to keep the diploid, coordinating cell cycle stages of donors and recipients to increase nuclear transplant efficiency, etc.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12528
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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转植基因鱼的核移植研究.赵浩斌[d].中国科学院水生生物研究所,1999.20-25
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