磁小体介导制备转基因猪模型  

Producing Transgenic Pig by Bacteria Magnetic Particles

作　　者：陈克研[1] 陈振文[2] 董婉维[1] 郑志红[1] 

机构地区：[1]中国医科大学实验动物部,沈阳110032 [2]首都医科大学基础医学院实验动物学系,北京100069

出　　处：《实验动物与比较医学》2015年第4期288-293,共6页Laboratory Animal and Comparative Medicine

基　　金：国家自然青年科学基金项目（31201758）,中国博士后基金（2012M521919）

摘　　要：目的以磁小体（BMP）和聚乙烯亚胺（PEI）为载体，通过睾丸注射精子介导法建立转基因猪模型。方法将实验室保存的脂蛋白磷脂酶A2（Lp．PLA2）绿色荧光蛋白（GFP）质粒与BMP和PEI混匀，制备BMP—PEI-PLA2-GFP混合物，采用睾丸多点注射法接种巴马小型猪，于接种后第2周、4周、6周至第20周采集精液，分别用RT—PCR和免疫荧光鉴定外源基因的表达情况；取阳性精液，对母猪实施人工授精；PCR及RT—PCR对所产仔猪进行鉴定。结果精液中GFP基因于接种后第4周首现并持续至第16周；荧光显微镜检查可观察到GFP蛋白主要表达于精子项体后区及精子尾颈部；经人工授精所产21头仔猪中，9头猪用PCR可检测GFP片段，用RT—PCR检测PLA2表达，该9头猪的表达量明显高于其它猪，总阳性率达42．86％。结论BMP和PEI结合可作为良好的携带外源基因的非病毒性载体，用该载体通过精子介导法可以达到迅速制备转基因猪的目的，其成本低、方法简单、便捷。Objective To establish sperm-mediated transgenic pigs models by intratestis injection with bacteria magnetic particles （BMP） and polyethylenimine （PEI） as a vector. Method The Lp-PLA2- GFP plasmid were mixed with BMP-PEI to BMP-PEI- PLA2-GFP, which were delivered into Bama Mini-pigs by intratestis injection. The sperm were collected in 2 weeks, 4 weeks, 6 weeks to 20 weeks after inoculation, and the GFP gene were detected by RT-PCR and immunofluorescence. The positive sperms will be transferred through artificial insemination to sows to establish transgenic pigs. The piglets were detected by RT-PCR to GFP gene and Real-time PCR to Lp-PLA2 gene. Results The GFP gene was first appearance in semen in 4 weeks after inoculation, and sustained through 16 weeks. The GFP protein mainly expressed in the sperm acrosome, sperm tail and neck area were observed by fluores- cence microscopy. Through artificial insemination, 21 piglets were born, 9 of which were found with GFP segments, and the expression of PLA2 was obviously higher than that of other pigs, with a total positive rate of 42.86%. Conclusion The BMP combined with PEI can serve as a good non viral vector carrying exogenous gene, and sperm-mediated method with the vector can achieve the purpose of rapid preparation of transgenic pigs, its cost is low, the method is simple and convenient.

关 键 词：猪 转基因 磁小体(BMP) 聚乙烯亚胺(PEI) 精子介导的基因转移 

分 类 号：Q95-33[生物学—动物学]