摘要
针对实验室中比较普遍的包装的慢病毒滴度不高的问题,我们对慢病毒包装过程中的几个关键步骤进行了优化。采用Fugene6同时转染携带GFP的质粒和包装慢病毒所必需的包装质粒进入293T细胞中。通过使用荧光显微观察GFP的表达亮度及流式细胞技术测量GFP阳性细胞的比例来测定病毒滴度。通过优化Fugene6和DNA的比例,发现当Fugene6和DNA的比例是3:1时获得的慢病毒的滴度最高;通过对转染后收获病毒的时间的比较,发现转染48小时后回收所得到的慢病毒滴度最高。
Lentivirus has been used widely as an exogenous gene delivery tool. However, it is relatively difficult to get high titer lentivirus. The purpose of this article was to modify two critical steps during the lentivirus packaging procedure in order to get high titer lentivirus. The two critical steps were the ratio between transfection agent and target vector, and the lentivirus collecting time. Fugene6 was used as the transfection agent. Plasmid car- rying green fluorescent protein （GFP） sequence was used as the target vector. Packaging plasmid, envelope plasmid and target vector were co-tranfected into the 293T ceils. The GFP positive 293T cells were visualize by fluorescence microscope. The percentage of GFP positive cells were analyzed by flow cytometry. We found that when the ratio of Fugene6 and target DNA was 3：1, the lentivirus titer was the highest comparing with other options. The lentivirus collected at 48 hours after transfection had the highest infection property.
出处
《中国细胞生物学学报》
CAS
CSCD
2011年第4期385-390,共6页
Chinese Journal of Cell Biology
基金
上海市大学生创新计划(No.1500107029)
河北省医学科学研究重点课题计划(No.20100313)~~
