An optimized method for high-titer lentivirus preparations without ultracentrifugation.
An optimized method for high-titer lentivirus preparations without ultracentrifugation.
复制标题
一种无需超速离心的高滴度慢病毒制备的优化方法
DOI：
10.1038/srep13875
复制
发表时间：
2015-09-08
影响因子：
4.6
通讯作者：
Zhang C
中科院分区：
文献类型：
--
作者：
Jiang W;Hua R;Wei M;Li C;Qiu Z;Yang X;Zhang C
慢病毒技术已被证明是在分裂和非分裂细胞中表达外源基因的有力工具。目前，用于产生高滴度慢病毒的大多数方案需要超离心，这可能是实验室中常规操作的仪器屏障。系统研究了相对离心力（RCF）对慢病毒浓缩效率的影响，发现蔗糖梯度离心（≤ 10，000 g）可稳定地产生高滴度病毒（达2× 108 TU/ml）。最佳蔗糖浓度为10%，功能性病毒的回收率大于80%。当病毒在4 ℃下储存（τ = 1.3天）或经历多次冻融循环（τ= 1.1轮）时，浓缩和未浓缩的慢病毒的感染效率迅速降低。总之，我们描述了一种高效且易于操作的高滴度慢病毒纯化方案。
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.