中文摘要：

【目的】精子介导的转基因技术简单易行,其作为制备转基因动物的一种方法已经被很多科学家所认同。但是不同实验室的研究结果显示其稳定性和重复性较低,转基因效率差异极大。现将主要探讨导致这种现象的原因。【方法】小鼠精子获能后分别与0、15、30和300nmol·L-1的Cy-3标记DNA（Cy-3-DNA）在37℃共孵育30 min。其后,血细胞计数板检测小鼠精子的活率;共孵育精子进行直接涂片,DPBS洗涤后涂片,DnaseI消化后涂片,精子涂片置于荧光显微镜下,记录视野中的精子总数及显示Cy-3信号的精子数,用于统计精子结合及内化DNA的效率。精子与300 nmol·L-1的Cy-3-DNA共孵育后,以50μmol·L-1的P4诱导精子顶体反应,统计顶体反应前后显示Cy-3信号的精子比例。精子分别与15和300 nmol·L-1 Cy-3-DNA共孵育后,精子进行体外受精,在荧光镜下观察合子期受精卵中Cy-3-DNA的存在位置。按照优化后的条件,精子分别与0、15和300 nmol·L-1 pEGFP-C1质粒共孵育后进行体外受精,制备转基因胚胎。比较试验组和对照组的受精率和发育效率,采用PCR和RT-PCR方法检测囊胚期外源基因整合和表达情况。【结果】获能后的精子分别与0、3、15、30和300 nmol·L-1的Cy-3-DNA共孵育后,精子的活率分别为82.21%、73.63%、77.38%、76.33%和77.80%;洗涤前精子结合DNA的效率分别为76%、94%、99%和100%,15、30和300 nmol·L-1组精子结合率均显著高于3 nmol·L-1组（P〈0.05）。洗涤后精子结合DNA的效率,分别为45%、66%、84%和87%,消化后精子内化DNA的效率分别为44%、56%、71%和76%,并且洗涤后精子结合及消化后精子内化DNA的效率都为300 nmol·L-1组和30 nmol·L-1组均显著高于其他两组,而15 nmol·L-1组显著高于3 nmol·L-1组（P〈0.05）;Image J对精子结合Cy-3-DNA的强度进行分析的结果表明,外源DNA的量随着外源DNA浓度的增加而显著增加（P〈0.01）。顶体反应后,精子顶?

英文摘要：

[Objective] Sperm mediated gene transfer technique is simple, it has been recognized by many scientists as a way to produce transgenic animals, however, the results from different laboratories showed poor stability and reproducibility, transgenic efficiency is also significantly different. The aim of this study is to clarify the main factors causing this phenomenon. [Method] Capacitated sperms were incubated with 0, 15, 30 and 300 nmol.Ll Cy-3 labeled DNA （Cy-3-DNA） at 37℃ for 30 min. After incubation, sperms viabilities were detected by CBC board. Sperms incubated by DNA were used to direct smeared, smeared after washing by DPBS and smeared after digested by Dnase I, sperm smears were placed under fluorescent microscope. The total number of sperms and the number of sperms which show Cy-3 signal in the vision were recorded, the records were used for the efficiency statistics of sperms binding and uptaking exogenous DNA. 501~mol-L1 P4 were used for inducing sperms acrosome reaction which were incubated with 300 nmol.L-1 Cy-3-DNA. The proportion of sperms showing Cy-3 signals were checked before and after the acrosome reaction. Sperms incubated with 15 and 300 nmol.L-1 Cy-3-DNA were used for IVF, the presence of Cy-3-DNA in the zygotes was detected under fluorescence microscopy. According to the optimized conditions for these experiments, sperms incubated with 0, 15 and 300 nmol.L1 pEGFP-C1 were used for getting transgenic embryos, and oocyte fertilization rate and embryonic development rate in experimental group and control group were compared, PCR and RT-PCR were used to detect the presence and expression of pEGFP in the blastocyst. [Result] Sperms after capacitation were incubated with 0, 30, 15, 30 and 300 nmol.L1 Cy-3-DNA, the sperm viability was 82.21%, 73.63%, 77.38%, 76.33% and 77.80%, respectively. The rate of positive sperms was 76%, 94%, 99% and 100% before washing, and that in 15, 30 and 300 nmol＇Ll treatment groups had more efficiency than 3 nmol＇L1 （P〈0.05） group. The rate of positive