【作者】 张鑫；

【导师】 尹逊河； 苗向阳；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 在过去的近30年中,转基因技术在哺乳动物基因表达方面的研究应用,已经成为实验生物学及应用生物学领域最为显著的进展之一。传统的制作转基因动物方法有显微注射法、逆转录病毒感染法和胚胎干细胞法等,但每种方法都有其缺陷,限制了其在今后转基因动物研究中的广泛应用。通过睾丸注射法对体内生殖细胞进行外源基因转染是近些年来发展起来的一种进行转基因动物研究的新方法。由于其操作简便、成本低廉、效率较高且适于大量生产转基因动物的优点而越来越受到关注。但由于该方法起步较晚,目前还存在许多问题。因此,对该方法进行研究和完善,对今后制作和生产转基因动物,培育抗病动物品种以及通过建立转基因动物生物反应器生产珍贵蛋白等都具有重要意义。本研究共分三部分:第一部分:小鼠精子形成各阶段转基因效率的研究本试验对小鼠体内生殖细胞进行外源基因转染,研究精子形成过程中制作转基因小鼠的效率。首先运用睾丸注射法将被脂质体包裹的绿色荧光蛋白表达载体(pIRES2-EGFP)注射到公鼠睾丸及附睾内,然后根据精子形成不同阶段,分别于注射后7d、16d、30d和42d与发情母鼠合笼,利用PCR和Southern印迹方法对新生小鼠进行基因组DNA检测。在各阶段所得新生小鼠中PCR阳性率分别为6.82%、0、56.86%和、42.86%,Southern印迹检测阳性率为分别为6.82%、0、47.06%、34.69%,经活体荧光成像系统及荧光显微镜分析,转基因小鼠呈现绿色荧光表达。通过比较精子生成各阶段转基因效率高低,为以后通过用睾丸内注射法转染雄性生殖细胞高效制作转基因动物提供了理论依据。第二部分:睾丸注射法在不同生精周期制作转基因小鼠效率的研究运用睾丸注射法将被脂质体包裹的绿色荧光蛋白表达载体(pIRES2-EGFP)注射到公鼠睾丸内,然后根据小鼠精子的形成周期,分别于注射后第42d、84d、126d、168d和210d即注射后连续5个周期内与正常发情母鼠合笼,利用PCR和Southern印迹方法对新生小鼠进行基因组DNA的跟踪检测。在各阶段所得新生小鼠中PCR阳性率分别为36.36%、17.78%、12.50%、10.87%、4.65%,Southern印迹检测阳性率为分别为31.82%、13.33%、8.33%、6.52%、2.33%,经活体荧光成像系统及荧光显微镜分析,转基因小鼠呈现绿色荧光表达。通过比较不同精子生成周期的转基因效率,初步表明精原干细胞可以长时间携带外源基因,但其效率随时间的延长而明显降低,这为今后通过用睾丸内注射法转染精原干细胞制作转基因动物提供了基础理论。第三部分:睾丸注射法建立抗菌肽Thanatin转基因小鼠的初步研究人工合成抗菌肽Thanatin基因的三条片段,利用重叠延伸PCR扩增技术得到完整的Thanatin基因,通过分子克隆方法构建出pIRES2-EGFP-Thanatin重组表达载体。然后与脂质体混合,采用睾丸打点注射将新构建的抗菌肽基因注入小鼠睾丸内,共注射公鼠5只。6周后与雌鼠交配,对F1代新生小鼠进行断尾,提取尾尖基因组DNA,利用PCR和Southern印迹方法对其进行检测,从检测阳性小鼠中随机挑选公、母鼠各3只分别与正常小鼠交配,用PCR方法对F2代进行检测。结果得到的52只F1代鼠中PCR检测阳性率为38.46%,Southern印迹检测阳性率为30.77%;转基因阳性小鼠所生55只F2代中,PCR检测阳性率36.36%;在活体荧光成像系统下转基因小鼠呈现绿色荧光表达。通过睾丸注射法使Thanatin基因在小鼠的基因组中得到整合,为今后进一步研究抗菌肽的作用机理、培育抗病动物品种以及今后通过建立转基因动物生物反应器进行抗菌肽的大量生产打下了基础。更多还原

【Abstract】 In the past 30 years, the research and application of transgenic technology in gene expression of animal had became noticeable advancement in experimental biology and applied biology. Microinjection,retrovirus mediated method and embryonic stem cells method were all the traditional methods of producing transgenic animals. But these methods had defects and limited application in the research of transgenic animals in the future. Using intratesticular injection is a new method to product transgenic animal recently. There are many merits of intratesticular injection, simple operation, low cost, higher efficiency and suitable producting transgenic animal. There are important significance to study this method for producting transgenic animal, disease-resistant animal breeding and production of valuable proteinum through transgenic animals.Two parts were included in the studies:PartⅠ: Study of the Transgenic Efficiency in Difference Spermiogenesis Stages in MiceIn this experiment exogenous gene was transferred into germ cells of male mice, in order to study efficiency of transgenic mice in different spermiogenesis stages.The green fluorescence protein expression plasmid(pIRES2-EGFP)and liposomes were mixed and injected into male mouse testis and epididymis. Then the intratesticular mice were mated with female at day 7,16,30 and 42th after infection. Polymerase chain reaction(PCR) and southern blot were applied to identify transgenic mice. The positive ratio was 6.82%、0、56.86%、42.86% by PCR analysis and 6.82%、0、47.06%、34.69% by southern blot analysis . The transgenic mice showed green fluorescence in fluorescence imager and fluorescent microscope under EGFP excitation light (488nm). Through comparing efficiency of transgenic mice in different spermiogenesis stages, it could provided an important theoretic foundation for efficient production of transgenic animals by testis mediated gene transfer.PartⅡ:Study of Transgenic Efficiency by Intratesticular Injection in Different Spermatogenic Cycles in MiceThe green fluorescence protein expression plasmid(pIRES2-EGFP)and liposomes were mixed and injected into male mouse testis. Then the intratesticular mice were mated with female at day 42,84,126,168 and 210th after injection. Polymerase chain reaction (PCR) and southern blot were applied to identify transgenic mice. The positive ratio was 6.36%、17.78%、12.50%、10.87%、4.65% by PCR analysis and 31.82%、13.33%、8.33%、6.52%、2.33% by southern blot analysis . The transgenic mice showed green fluorescence in fluorescence imager and fluorescent microscope under EGFP excitation light (488nm). Through comparing the efficiency of transgenic mice in different spermatogenic cycles, it shows that the spermatogonial stem cells could carry exogenous genes for a long time but the efficiency decreases with time. This provided an important theoretic foundation for efficient production of transgenic animals by testis mediated gene transfer.PartⅢ:Preliminary Research on Production of Transgenic Mice Carrying Thanatin GeneThanatin gene was constructed by overlap extension PCR and inserted into pIRES2-EGFP vector. Then recombinant plasmid pIRES2–EGFP -Thanatin and liposome were mixed and injected into male mouse testis. Six weeks after injection, male mice mated with female mice. Genomic DNA was extracted from F1 pups tail.Polymerase chain reaction(PCR) and southern blot were applied to identify transgenic mice. Six mice were selected from F1 positive mice, then mated with normal mice and detected by PCR. Fifty two F1 pups were born and the positive ratio was respectively 38.46% and 30.77% by PCR and southern blot analysis. In F2 pups, the positive ratio was 36.36%. The result showed that thanatin gene was integrated into mice genomic DNA. Expression of green fluorescence protein in transgenic mice was observed in fluorescence imager. In the present study, establishment of transgenic mice carrying thanatin gene is attractively valuable and prospective in application. And this provides a basis for disease-resistant animal breeding and production of antibacterial peptides through transgenic animals.更多还原