细菌磁小体介导转基因猪研究

Transgenic animals have played an important role in human disease research, but most transgenic animal production methods exist shortcomings in either technicdifficulties, gene modify low efficiency and high costs so far. Intratesticular injection is a new transgenic animal method, which has the merits of easy to operate, low cost and high efficiency, which is attracting more attention to researchers. The disadvantage of intratesticular injection is its unstable transgene output. Bacteria magnetic particles (BMP) are composed of iron oxide enclosed within biomembrane. They can combine with PEI covalentely through the steroids and proteins connected to the biomembranes. As a result, BMPs, PEI and the foreign gene segments connected to PEI will form into a complex, which prevent the degradation of DNA from DNase. At the same time, the complex will move along with magnetic field towards the target,which could be a suitable candidate vector for introducing medicines into target tissues in vivo. Our study show that afer mice HMGR gene specific shRNA plasmid segment can combine with BMP-PEI and be introduced into mice testis, transgenic mice are successfully and the efficacy is superior to conventional method. Accordingly, we design the Mini-pig transgenic assay by intratesticular injection to examine if BMP mediated trnasgene is applicable to laboratory animals bigger than mice. In this study, the green fluorescent protein (GFP) marked BMP-PEI-GFP complex will be delivered into Bama Mini-pigs by intratesticular injection to produce transgenic positive sperms. And then, pigs will mate naturally or, the positive sperms will be transferred through artificial insemination to sows to established transgenic pigs. This study will not only set up a new transgenic methods of Mini-pigs, but also provide important technical support to larger animals transgenic research. Furthermore, it is helpful to explore the integration mechanism of BMP-PEI mediated transgene. In addtion, establishing transgenic pig model is important for human disease research and provide support to medicine industry development.

目前，常用的转基因动物制作方法大多存在技术难度大、转基因效率低及成本高等缺陷，而睾丸注射转基因法由于操作简便、成本低、效率高而倍受关注，但稳定性有待提高。研究表明，聚乙烯亚胺(PEI)与磁小体 (BMP) 结合，通过外加磁场进行靶向基因导入，可有效提高转基因效率和基因表达水平。本课题组前期在合成BMPs-PEI-HMGR的基础上，通过睾丸注射法导入小鼠，获得高效率的转基因阳性小鼠。据此，我们设计小型猪睾丸注射磁小体转基因方法。本研究拟通过制备BMP-PEI-GFP复合物，经睾丸注射导入巴马小型猪后外加磁场干预，从而产生转基因阳性精子，再通过本交或人工授精技术建立转基因猪。该研究不但建立了一种新型的小型猪转基因制作方法，为其它大动物转基因研究提供重要的技术支持；同时还在一定程度上阐明BMP-PEI介导的基因与细胞的整合机制。转基因猪动物模型的建立，将为人类疾病研究和医药产业发展提供材料支撑。

目前，常用的转基因动物制作方法大多存在技术难度大、转基因效率低及成本高等缺陷，而睾丸注射转基因法由于操作简便、成本低、效率高而倍受关注，但稳定性有待提高。本研究利用磁小体（BMP）的趋磁性和较强的吸附DNA能力，结合聚乙烯亚胺（PEI）转染效率高且不易降解等特点，制备BMP-PEI- PLA2-GFP混合物；建立巴马小型猪睾丸注射导入外源基因的方法，经检测于注射后第2周起对精液DNA进行PCR扩增，结果GFP基因于第4周首现并持续至第16周。荧光镜检发现，注射后第7周为GFP蛋白表达的高峰期，荧光镜检可观察到表达GFP蛋白的精子，GFP蛋白主要表达于精子顶体后区及精子尾颈部。注射2周后精子的畸形率达到33%左右，高于对照猪精子畸形率的21%，而后呈下降趋势，于第6周降至23%左右，并趋于稳定。吖啶橙染色检测精子DNA双链完整性良好。在术后第4、8周取猪睾丸做冰冻切片并于荧光显微镜下观察GFP蛋白的表达。结果转基因复合物在注射后第4周与第8周均可观察到GFP蛋白表达，其主要位于曲细精管基膜或者管间。本实验通过人工授精8头母猪，其中有6头怀孕，共产仔猪44头，分别提取DNA，进行PCR检测，结果18头呈阳性；荧光定量PCR检测结果显示这18头仔猪PLA2的表达量均高于其它猪，Western-blot对PLA-2蛋白检测同样在这18头仔猪中有高表达的PLA2蛋白，总阳性率为40.91%；进一步原代培养转基因猪猪尾成纤维细胞，置荧光显微镜下观察，结果这18头猪的猪尾成纤维细胞均能观察到GFP的表达，从而确认磁小体介导的转基因猪的成功构建。综上，本研究证实磁小体与聚乙烯亚胺结合可作为良好的携带外源基因的非病毒性载体；用该载体通过睾丸多点注射即可实现转基因的目的，其成本低、方法简单、便捷；通过睾丸注射可将外源基因DNA吸附于精子上，其持续时间较长，通过体外人工授精可以达到迅速制备转基因猪的目的。