An optimized method for high-titer lentivirus preparations without ultracentrifugation

Scientific Reports
Wei JiangChen Zhang

Abstract

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.

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Citations

Nov 9, 2016·Proceedings of the National Academy of Sciences of the United States of America·Jihong GongJiajie Diao
May 24, 2017·Antimicrobial Agents and Chemotherapy·Eric Fleta-SorianoAndreas Meyerhans
Oct 16, 2018·Biotechnology & Genetic Engineering Reviews·Andrew Octavian Sasmita
Oct 31, 2018·The Journal of Immunology : Official Journal of the American Association of Immunologists·João V S OrmondeJoaquín Madrenas
Oct 27, 2018·Journal of Cellular Biochemistry·Zohreh BolandiHossein Ghanbarian
Apr 17, 2019·RNA·Mateusz WawroAneta Kasza
Jul 18, 2019·Critical Reviews in Clinical Laboratory Sciences·Mansour PoorebrahimAngel Cid-Arregui
Feb 18, 2020·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Anna TanakaAtsushi Miyajima
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Jul 2, 2020·Experimental Neurobiology·Tae-Young LeeHaeyoung Suh-Kim
Dec 2, 2017·Scientific Reports·Mohammed Hakim JafferaliEinar Hallberg
Nov 20, 2019·International Journal of Molecular Sciences·Nicolas JungSabrina Bréchard
Jul 28, 2020·Journal of the American Society of Nephrology : JASN·Thati MadhusudhanBerend Isermann
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Methods Mentioned

BETA
transfection
transfections
PCR

Software Mentioned

FlowJo
Summit
Clampfit

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