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The Optimized Protocol of Making High Titer Lentivirus by Using Fugene6
Lin Fan1, Zhi-Zhao Ma2, Shan-E Gao1, Jian-Wei Lu3, Si-Guang Li1, Xiao-Jing Pan1*
1Tongji University Medical School, Stem Cell Research Center, Shanghai 200092, China; 2Hebei Medical University Second Hospital,Neurosurgery, Shijiazhuang 050000, China; 3Tongji University Medical School Embryonic Stem Cel
Abstract: 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 carrying green fluorescent protein (GFP) sequence was used as the target vector. Packaging plasmid， envelope plasmid and target vector were co-tranfected into the 293T cells. 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.
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