利用精子载体法制备转基因小鼠疾病模型的初步研究

发布时间：2018-03-04 11:44

本文选题：精子载体法　切入点：转基因动物　出处：《莱阳农学院》2006年硕士论文　论文类型：学位论文

【摘要】：精子介导基因转移是以精子为载体,在受精时将外源目的基因导入卵母细胞,是目前转基因动物研究中简单而高效的方法学之一。本研究以ICR小白鼠为试验动物,用精子载体法制备转基因小鼠模型,并根据得到的结果分析讨论精子载体法制备转基因动物的可行性及优缺点。本文进行了如下研究: 外源DNA转染小鼠精子的研究:利用DIG末端标记技术和免疫组化技术研究小鼠精子转染外源DNA的效率。试验结果发现,不同小鼠个体精子转染阳性率有明显差异,平均为13%左右。小鼠精子具有自发结合外源DNA能力,结合部位集中在精子顶体后躯,结合的外源DNA首先“挂”在精子膜表面,继而被内化转运到细胞内,但并非所有的结合DNA都被内化。精子介导法制备转基因小鼠的试验研究:利用考马斯亮蓝染色技术评价小鼠精子顶体反应发生的情况,选择合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP并获能的小鼠精子对成熟卵母细胞进行体外受精,受精卵进行体外培养,荧光显微镜下观察胚胎,结果表达GFP的阳性率为4.7%。试验结果证明了精子介导法制备转基因小鼠的可行性。乙肝病毒结合蛋白表达载体的构建:利用PCR和基因重组技术构建了HBV结合蛋白表达载体,经过酶切鉴定和基因测序证实了载体序列的正确性,并作为外源DNA准备用于转染小鼠精子制备动物疾病模型。本研究初步建立了利用精子载体法制备转基因小鼠的平台,为今后制备动物疾病模型奠定了基础。但没有进行HBV结合蛋白基因的转染,今后还需对本试验作进一步的研究。
[Abstract]:Sperm mediated gene transfer to spermatozoa during fertilization, the exogenous gene into oocytes, transgenic animal research is currently one of the simple and efficient methodology. In this study, ICR mice as experimental animal, transgenic mice model using sperm vector method, and discussed the feasibility and advantages and disadvantages sperm carrier preparation of transgenic animal according to the results.
The following study is carried out in this paper.
Study of exogenous DNA transfection of mouse spermatozoa: efficiency by DIG end labeling and immunohistochemistry study of mouse spermatozoa transfected with exogenous DNA. The test results show that the obvious difference between the positive rate of different individual mouse sperm transfection, the average is about 13%. The mouse sperm has spontaneous binding of exogenous DNA, the binding site focused on sperm acrosome the hindquarters, exogenous DNA with first "hang" on the surface of the sperm membrane, then transport is internalized into cells, but not all of the combination of DNA have been internalized.
Study on Preparation of transgenic mouse sperm mediated method: Using Coomassie brilliant blue staining technique to evaluate mouse sperm acrosome reaction, select the appropriate in vitro fertilization in vitro. The semen of mice, the mice sperm in vitro transfection of GFP and capacitation in vitro fertilization of mature oocytes, fertilized eggs were cultured in vitro. The embryo was observed under fluorescence microscope, the positive expression rate of GFP proved the feasibility of preparation of transgenic mouse sperm mediated by 4.7%. test results.
Hepatitis B virus binding protein expression vector construction: to construct the HBV binding protein expression vector using PCR and gene recombination technique. After enzyme digestion and sequencing confirmed the correctness of the vector sequence, and as exogenous DNA prepared for transfection of mouse sperm preparation of animal disease model.
In this study, we initially established a platform for preparing transgenic mice by sperm carrier method, which laid the foundation for the preparation of animal disease models. However, no HBV binding protein gene was transfected. Further research is needed in the future.

【学位授予单位】：莱阳农学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332

【引证文献】