慢病毒载体转染犬睾丸生殖细胞  

Transfection Efficiency of Lentiviral Vector-based SMGT for Dog Testicular Germ Cells in vivo

作　　者：王洪军[1] 马啸[2] 赵明[1] 杜小燕[2] 李益琛[2] 陈振文[2] 

机构地区：[1]内蒙古民族大学附属医院,通辽028000 [2]首都医科大学基础医学院,北京100069

出　　处：《实验动物科学》2013年第1期6-10,共5页Laboratory Animal Science

基　　金：国家自然基金面上项目(30970420;31172168);国家科技支撑项目(2011BAI15B01-32)

摘　　要：目的观察慢病毒载体(Lentiviral vector,LV)对犬生殖细胞的转染,为基于LV的精子介导法制备转基因动物提供依据。方法采用睾丸打点注射法,向比格犬两侧睾丸分别注入滴度为5×108、1×108、2×107TU/mL的慢病毒液组成3个剂量组,每点注射量为每只犬0.2 mL。于注射后第2周开始采集精液,通过精液DNA的PCR检测和精子荧光镜检评估绿色荧光蛋白(green fluorescent protein,GFP)基因的整合及表达。结果 1)在高剂量组,整合并表达GFP基因的精子于第4周首现并持续至第17周;在中、低剂量组于第5周首现,分别持续到第14周、12周。2)GFP表达的高峰期在注射后第7周～10周。3)GFP表达于整个精子,以顶体后区和精子尾颈部表达最强。4)注射后第2周～6周,中剂量组犬采精困难,高低剂量组犬精子畸形率有上升的趋势。5)在GFP表达的高峰期,绿色荧光精子的百分率在高、中、低剂量组分别为43.33%、35.53%和4.55%,具有明显的差异。结论基于LV的精子介导转基因法(Sperm-mediated gene transfer,SMGT)可成功获得转基因犬精子,转基因阳性率、表达的强度与持续时间与慢病毒滴度相关。Objective To observe the transfection efficiency of lentiviral vector for dog testicular germ cells so as to provide the foundation for generating the transgenic animal by lentiviral vector-based sperm-mediated gene transfer. Method LV-GFP with the titers 5×10^8、1×10^8、2×10^7TU/ mL was injected respectively into the bilateral testis of Beagle dogs in high, medium and low-dose group at a dose of 2 ml/dog. Sperm-collecting was set about from the second week after the testicular injection. The integration and expression of GFP gene were assessed by PCR tests of semen DNA and sperm detection with fluorescence microscope. Results 1） In high-dose group the integration and expression of GFP gene firstly appeared at the 4th week and maintained till the 17th week, and it appeared at the 5th week in the medium and low-dose group and lasted till to the 14th and 12th week respectively; 2） The peak level of GFP expression occurred in 7 to 10 weeks after the testicular injection; 3） GFP expressed in the entire sperm, but expressed highly in the acrosomal back zone and the neck section of sperm tail; 4） There was ascent trend of dog sperm deformity rate in high and low-dose group during 2 weeks to 6 weeks after the injection; 5） During the peak period of GFP expression, the percentage of GFP sperm in high, medium and low-dose group was respectively 43.33% , 35.53% , 4.55% , with a significant difference. Conclusion The lentiviral vector- based SMGT can successfully generate transgenic dog sperm, and the transgenic rate, the intensity and the duration of transgenic expression were related to the titer of lentiviral vector.

关 键 词：比格犬 慢病毒载体 生殖细胞 

分 类 号：S829.2[农业科学—畜牧学]