An optimized method for high-titer lentivirus preparations without ultracentrifugation.
Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2×10(8) TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ≈1.3 days) or subjected to multiple freeze-thaw cycles (τ=1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.
State Key Laboratory of Membrane Biology, School of Life Sciences; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China.|Key Laboratory of Cognitive Science, Laboratory of Membrane Ion Channels and Medicine, South-Central University for Nationalities, Wuhan 430074, China.
Wei
W
Jiang
State Key Laboratory of Membrane Biology, School of Life Sciences; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China.|Key Laboratory of Cognitive Science, Laboratory of Membrane Ion Channels and Medicine, South-Central University for Nationalities, Wuhan 430074, China.
Rui
R
Hua
State Key Laboratory of Membrane Biology, School of Life Sciences; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China.|Key Laboratory of Cognitive Science, Laboratory of Membrane Ion Channels and Medicine, South-Central University for Nationalities, Wuhan 430074, China.
Mengping
M
Wei
Key Laboratory of Cognitive Science, Laboratory of Membrane Ion Channels and Medicine, South-Central University for Nationalities, Wuhan 430074, China.
Chenhong
C
Li
Laboratory of Molecular Basis of Neural Plasticity, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031, China.
Zilong
Z
Qiu
Key Laboratory of Cognitive Science, Laboratory of Membrane Ion Channels and Medicine, South-Central University for Nationalities, Wuhan 430074, China.
Xiaofei
X
Yang
State Key Laboratory of Membrane Biology, School of Life Sciences; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China.
Chen
C
Zhang
D000818:Animals; D001709:Biotechnology / Q000379:methods; D002460:Cell Line; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D009474:Neurons / Q000378:metabolism; D014161:Transduction, Genetic; D014461:Ultracentrifugation; D019562:Viral Load; D014779:Virus Replication