摘要
慢病毒介导法是最有前途的转基因动物生产方法之一,高滴度慢病毒颗粒的包装是慢病毒转基因动物技术的关键。本研究应用含有增强型绿色荧光蛋白基因(eGFP)的第3代慢病毒载体系统,用脂质体转染法将慢病毒系统4质粒共转染293T包装细胞,培养48～72h收集病毒上清液,通过超速离心进行浓缩,采用批量快速测定法(LaSRT)测定病毒滴度。结果显示,用脂质体转染法包装的慢病毒能成功地感染293T细胞,经检测病毒滴度达到5×108IU/mL以上,初步建成了高滴度慢病毒包装平台,为慢病毒介导制作转基因动物奠定了良好的研究基础。
Lentivirus-mediated method is one of the most promising methods of producing transgenic animals, high-titer lentiviral particles packaging is the key of lentivirus-mediated transgenic animal technology. In this study, 293T cells were co-transfected with four plasmids, which are the 3^rd generation of lentiviral vector systems containing enhanced green fluorescent protein gene （eGFP）, using the Lipofectamine 2000 mediated transfection method. Virus supernate was collected after 48-72 hours, and then concentrated by ultracentrifuge. Large-scale real-time titration （LaSRT） was applied to test the titer of virus. The results showed that 293T cells could be successfully infected by the virus packaged using Lipofectamine-mediated transfection, as evidenced of that the virus titer reached higher than 5×10^8 IU/mL, indicating that a high-titer lentiviral packaging platform was preliminary estabilished. This result will be beneficial for the further study of producing transgenic animal by lentivirus-mediated method.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2009年第2期326-330,共5页
Genomics and Applied Biology
基金
国家863计划(2007AA100505)
广西青年科学基金(桂科青0991002)
广西亚热带生物资源保护利用重点实验室开放课题(SB0702)资助
关键词
慢病毒
病毒包装
转基因动物
Lentivirus, Virus packageing, Transgenic animals
