【作者】 宋伟杰；

【导师】 李和平； 李善刚；

【作者基本信息】 东北林业大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 供体细胞在体外培养的时间和培养代数对体细胞核移植效率有重要影响。本研究通过对5-10低代数组和20-25高代数组的兔成纤维细胞进行核移植研究,结果发现两个实验组供体细胞之间的核移植的效率存有差异。通过对供体细胞进行核移植后的重构胚胎的融合率、卵裂率、囊胚率以及体内发育的比较,发现低代数相对来说效率较高,但是高代数组也能获得正常的克隆后代。本研究验证了兔不同代数体外培养细胞对克隆效率的影响,为制备基因打靶兔提供了理论依据和时间窗口。体细胞核移植已经成为制备转基因动物的常用技术,体细胞的转染效率已经成为制约转基因效率的重要因素。本实验通过脂质体转染体外培养的成纤维细胞,转染后按1：40和1：80两个密度进行稀释,生长至30%-40%汇合度时加入G418进行筛选,最终共获得细胞克隆41个,其中绿色荧光蛋白(GFP)阳性细胞克隆12个,两种稀释密度均可获得单细胞长满形成的GFP克隆,但低密度的效率更高,1：80可作为细胞筛选的适合密度。实验获得的结果为提高细胞转染效率和转基因动物制作的成功率提供了参考。胚胎冷冻保存已经成为动物保种,实验动物胚胎运输的一个必不可少的有效工具。玻璃化冷冻是一种快速冷冻方法,不需要特殊的设备。本研究将筛选出带有绿色荧光的胚胎应用玻璃化方法进行冷冻并将非绿色荧光的胚胎筛选出来后也进行玻璃化冷冻。将冷冻复苏后形态完整的胚胎进行体外培养,有47%的非GFP胚胎发育至囊胚。将复苏的GFP转基因桑椹胚移植到受体代孕母兔体内,21%的胚胎发育为胎儿。最终,通过玻璃化冷冻获得了4个转基因后代仔兔,并已经成活四个月以上。结果表明,GFP胚胎可以成功的应用玻璃化冷冻保存,并且应用荧光显微镜进行筛选能显著提高转基因胚胎保存的效率。更多还原

【Abstract】 The efficiency of somatic nuclear transfer was significantly influenced by in vitro culture time and passages of donor cells. We carried on nuclear transfer with 5-10 low passage and 20-25 high passage of rabbit fibroblasts,, and found that the efficiency of donor nuclei are different between two experimental groups. Through comparing the rates of fusion, cleavage, blastocyst development and in vivo development of reconstructed embryos, we found that low passage of donor cell was more effective than high one, but normal cloned offspring could be prouduced from high passage of cells. This study confirms the influence of different passage cells of in vitro culture on the efficiency of rabbit nuclear transfer, and provides experimental basis and time window for gene targeting in rabbit.Somatic cell nuclear transfer has become the common techniques of producing transgenic animal, but the genetic modification of donor cells is an important factor which restricts the efficiency of transgenic animal production. This experiment was carried on as follows, to transfect the fibroblasts first with Metafectene Easy, then dilute them according to 1:40 and 1:80 densities, respectively. Selective agent G418 was not added until the confluency of fibroblasts reached 30%-40%, and eventually 41 colonies were selected, among them 12 colonies contain GFP. GFP colonies could be obtained from both densities,but the efficiency of positive expression from low density of cells is a little bit higher, therefore the density of 1:80 was more appropriate to screen transfeced cell. The results could provide a reference for improving transfection efficiency of cells, and production of transgenic animals.Cryopreservation of embryos was a very useful and indispensable tool in animal breeding preservation and distribution of laboratory animals. Vitrification was a kind of quickly freezing method independent of specific machines. In the present experiment, GFP transgenic and non-GFP morulaes selected under the fluorescence microscopy were vitrified. After thawing, 47%of the non-GFP embryos survived, and could develop to blastocyst stage in vitro. When the thawed GFP transgenic morulaes were transferred into recipient rabbits,21%(10/47) of them could develop in full-term. Four transgenic rabbits from vitrified embryos grow up to 4 months old. The results suggested that Vitrification could be used to preserve transgenic rabbit strains and improved the efficiency of embryos stocking by filtering the transgenic embryos.更多还原