睾丸注射慢病毒载体制备转基因小鼠研究

Study on Generation of Transgenic Mice by Intratesticular Injection of Lentiviral Vector

目的探讨以睾丸注射法和慢病毒载体法相结合建立转基因动物的方法。方法用三质粒慢病毒载体系统和pSico-eGFP表达载体转染293TN细胞,包装慢病毒,转染后裂解细胞,收集携带绿色荧光蛋白(EGFP)基因的慢病毒。将30μL含1×TransDux和1%台盼蓝的病毒液2×107(U·m L^(-1))注射到成年C57BL/6J小鼠睾丸网内,根据小鼠精子形成周期,分别于注射后第7、15、30和42天与发情母鼠合笼。利用EGFP观察灯检测仔代转基因小鼠阳性率。结果该种方法制备转基因荧光小鼠成功,而且在注射后30天配种,产生的转基因小鼠阳性率最高,达35%。结论该方法简单,易于操作,能够有效制备转基因小鼠。

Objective To establish a method for generation of transgenic mice by intratesticular injection of lentiviral vector. Methods Three lentiviral vectors and p Sico-e GFP expression vector were transfected into293 TN cells,and virus particles expressing EGFP protein were harvested by cell lysis. 30 μL lentivirus media（2×10^7U·m L^-1）containing 1 x Trans Dux and 1% trypan blue was injected into rate testis of C57BL/6J mice. The intratesticular mice were mated with female at day 7,15,30 and 42 after injection. The transgenic mice were identified by EGFP excitation light（488nm）. Results The transgenic mice could be produced by intratesticular injection of lentiviral vector. The highest positive rate（35%）of offspring was at the 30 th day after injection.Conclusion Intratesticular injection is a simple and efficient method for production of transgenic mice.