【作者】 刘琼霞；

【导师】 陈铁桥； 燕海峰；

【作者基本信息】 湖南农业大学 ， 临床兽医， 2005， 硕士

【摘要】 20多年前有人提出利用转基因动物合成治疗蛋白。现在生产转基因动物的方法和在奶牛、绵羊、山羊的乳腺中特异性表达治疗蛋白取得了一定的进步。由于对转基因生物生产的人类生物药品需求的日益增加,发展利用转基因家鸡输卵管作为生物反应器在蛋清中生产外源蛋白显得尤为重要。在理论上转基因家禽较转基因哺乳动物具有不可比拟的优势,但由于家禽胚胎发育的特殊性,迄今为止家禽的转基因技术还落后于该技术在哺乳动物中的应用,为此我们进行了本研究。 研究目的:在SV40启动子的控制下,在家鸡囊胚细胞内导入和表达外源基因;对脂质体/DNA的比例及DNA-脂质体复合物的注射剂量进行优化,从而推究出脂质体介导GFP基因生产转基因嵌合体鸡的最佳技术路线,为转基因家鸡的生产提供一定的技术支持。 研究方法:本实验以白来航鸡种蛋为实验材料,体外分离鸡囊胚细胞并进行原代培养。分别用1、2、3、4、5ug的DNA和6u1脂质体(LIPOFECTAMINE 2000)的混合物体外转染来航鸡囊胚期原代细胞来优化质粒DNA(pEGFP-C1)和脂质体的最佳比例;用含1.5ug的DNA-脂质体复合物注射到新鲜种蛋的胚下腔;用荧光显微镜和PCR对胚胎和转基因鸡的各个组织器官进行检测。 实验结果:当脂质体(u1)与DNA(ug)的比例为2∶1时,其转染白来航鸡原代囊胚细胞12h的效果最好,转染贴壁细胞数达20%;不做任何处理、只开窗不注射、注射培养基、注射线性质粒DNA、注射脂质体-DNA复合物的种蛋孵化率分别为:91.67%、28.36%、13.67%、25.49%、30%;对转基因鸡胚进行荧光检测时,在5日龄鸡胚中观察到了极少量的绿色荧光,进行PCR检测时,检测到了5个阳性鸡胚,但是随着胚胎的发育,在后期没有出现阳性结果。 实验结论:脂质体/DNA复合物为2∶1时,转染贴壁白来航鸡原代囊胚细胞的效果最佳。对种蛋的不同处理方法,破坏了种蛋原来的结构,导致孵化率显著降低,且不同处理方法之间存在显著差异。对注射脂质体-DNA复合物到囊胚腔进行孵化的种蛋进行荧光检测和PeR检测,出现了阳性结果,说明外源DNA通过脂质体介导能导入鸡囊胚细胞。更多还原

【Abstract】 The potential of using transgenic animals for the synthesis of therapeutic proteins was suggested over twenty years ago. Considerable progress has been made in developing methods for the production of transgenic animals and specifically in the expression of therapeutic proteins in the mammary glands of cows, sheep and goats. Increased demand for the production of human biopharmaceuticals in transgenic organisms has led to an intensive effort to develop the hen as a bioreactor producing exogenous proteins in egg white via transgenesis. To date, however, robust methods for transgenic modification of the avian genome have lagged behind in mammalian systems. The positive features associated with the use of the chicken in terms of cost, speed of development of a production flock and potentially appropriate glycosylation of target proteins have led to significant advances in transgenic chicken models in the past few years. So we carry out this rearch.Research Objective: This study was to transfer and express foreign genes under control of the ovalbumin promoter in the BCs. We have used plasmid DNA (pEGFP-C1) in combination with cationic liposomes to transfect chicken BCs. To obtain higher efficiency in liposome-mediated gene transfer into chicken BCs cells. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy.Research Methods: The zygote laid by White Leghorn is used as expremental materials. In order to introduce exogenous DNA into gonads of chick embryos, stage X blastoderms of freshly laid and unincubated eggs were transfected by lipofection in vivo. Chicken BCs were isolated in vitro and cultured . Transfection efficiencies at different doses of DNA(1、 2、 3、 4、 5ug), liposome(6ul) were calculated under fluorescent microscopy using GFP(Green Fluorescent Protein) as a reporter gene. The liposome-DNA complex (1. 5ugDNA) were injected to the subgerminal cavity. We used microscopy and PCR to detect the GFP gene.更多还原