精子载体法制备FecB转基因太行山羊技术的研究

发布时间：2018-04-23 13:54

本文选题：精子载体法 + 脂质体　；参考：《河北农业大学》2011年硕士论文

【摘要】：精子介导的基因转移是指以精子作为外源基因的载体,在受精时将外源基因导入卵母细胞,使受精卵中含有外源基因,是目前转基因动物研究中简单而高效的方法之一。脂质体可用于转基因的研究,利用脂质体可以和细胞膜融合的特点,将外源基因送入细胞内部。本实验利用脂质体介导精子转染的方法,研究了精子在介导基因转移过程中精子与外源基因结合的影响因素,并对精子载体法制备FecB转基因太行山羊进行了初步研究。试验结果如下: 1.精子充分离心洗涤后,再与脂质体-DNA复合物混合,可显著提高脂质体介导精子转染外源DNA的效率。DNaseI消化前,洗涤组转染精子阳性率为38%,而未处理精液组精子阳性率为14%;DNaseI消化后,洗涤组转染精子阳性率为21%,而未处理精液组精子阳性率为5%,差异极显著(P 0.01)。 2.脂质体介导法可使外源DNA透过细胞膜内化进入精子胞质或胞核内,精子转染外源DNA用DNaseI消化后能检测到精子中外源DNA的存在。太行山羊精子与外源DNA混合培养后,用荧光原位杂交检测到18.3%的精子结合有外源DNA,脂质体Lipofectamine2000介导后比率增加至38.7%,两法的统计学分析存在显著性差异(P0.01)。 3.利用共培养法转染外源DNA,用DNaseI消化后,能检测到精子中外源DNA的存在,山羊精子具有自发的结合外源DNA的能力。 4.对18只太行山羊母羊进行诱导发情处理,精子与FecB基因共孵育后,对发情母羊进行输精,受孕率为51%,共产生后代12只,经过PCR检测,未发现阳性后代。
[Abstract]:Spermatozoa mediated gene transfer refers to the introduction of exogenous genes into oocytes during fertilization, which is one of the simple and efficient methods in the research of transgenic animals. Liposomes can be used in the study of transgene, and foreign genes can be transferred into the cells by the fusion of liposome and cell membrane. In this study, the factors affecting the binding of sperm to foreign genes in the process of sperm mediated gene transfer were studied by using liposome-mediated sperm transfection, and the preparation of FecB transgenic Taihang Sheep by sperm carrier method was studied preliminarily. The results are as follows: 1. After full centrifugation, spermatozoa were mixed with liposome-DNA complex. The efficiency of transfection of exogenous DNA into sperm mediated by liposome. DNase I was significantly improved. The positive rate of sperm transfection in washing group was 38 and that in untreated semen group was 14% after DNase I digestion. The positive rate of sperm transfection in washing group was 21 and that in untreated semen group was 5. The difference was significant (P 0.01). 2. Liposome-mediated method can make exogenous DNA enter the sperm cytoplasm or nucleus through the cell membrane internalization. After the sperm transfection of exogenous DNA is digested with DNaseI, the presence of DNaseI can be detected in the spermatozoa. After mixed culture of sperm and exogenous DNA in Taihang Mountain sheep, 18.3% of spermatozoa combined with exogenous DNA was detected by fluorescence in situ hybridization, and the ratio of liposome Lipofectamine2000 mediated was increased to 38.7%. There was a significant difference between the two methods in statistical analysis (P 0.01). 3. Exogenous DNA could be detected by co-culture and digested with DNaseI. Goat spermatozoa had the ability to combine exogenous DNA spontaneously. 4. After the estrus induction treatment was carried out in 18 estrous sheep of Taihang Mountain sheep, the estrus estrus was inseminated by sperm and FecB gene. The pregnancy rate was 51%, and 12 offspring were produced. After PCR test, no positive offspring were found.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：S827

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