An Optimized Method for High-titer Lentivirus Preparations Without Ultracentrifugation

Overview

Journal Sci Rep

Specialty Science

Date 2015 Sep 9

PMID 26348152

Citations 67

Authors

Wei Jiang

Rui Hua

Mengping Wei

Chenhong Li

Zilong Qiu

Xiaofei Yang

Chen Zhang

Affiliations

Soon will be listed here.

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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References

Nasri M, Karimi A, Farsani M . Production, purification and titration of a lentivirus-based vector for gene delivery purposes. Cytotechnology. 2014; 66(6):1031-8. PMC: 4235939. DOI: 10.1007/s10616-013-9652-5. View

Kafri T . Lentivirus vectors: difficulties and hopes before clinical trials. Curr Opin Mol Ther. 2001; 3(4):316-26. View

Zhang C, Wu B, Beglopoulos V, Wines-Samuelson M, Zhang D, Dragatsis I . Presenilins are essential for regulating neurotransmitter release. Nature. 2009; 460(7255):632-6. PMC: 2744588. DOI: 10.1038/nature08177. View

Reiser J . Production and concentration of pseudotyped HIV-1-based gene transfer vectors. Gene Ther. 2000; 7(11):910-3. DOI: 10.1038/sj.gt.3301188. View

Kumar P, Woon-Khiong C . Optimization of lentiviral vectors generation for biomedical and clinical research purposes: contemporary trends in technology development and applications. Curr Gene Ther. 2011; 11(2):144-53. DOI: 10.2174/156652311794940782. View

Wong L, Goodhead L, Prat C, Mitrophanous K, Kingsman S, Mazarakis N . Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications. Hum Gene Ther. 2006; 17(1):1-9. DOI: 10.1089/hum.2006.17.1. View

Al Yacoub N, Romanowska M, Haritonova N, Foerster J . Optimized production and concentration of lentiviral vectors containing large inserts. J Gene Med. 2007; 9(7):579-84. DOI: 10.1002/jgm.1052. View

Tang H, Kuhen K, Wong-Staal F . Lentivirus replication and regulation. Annu Rev Genet. 2000; 33:133-70. DOI: 10.1146/annurev.genet.33.1.133. View

Soler-Llavina G, Fuccillo M, Ko J, Sudhof T, Malenka R . The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo. Proc Natl Acad Sci U S A. 2011; 108(40):16502-9. PMC: 3189075. DOI: 10.1073/pnas.1114028108. View

10.

Yamada K, McCarty D, Madden V, Walsh C . Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer. Biotechniques. 2003; 34(5):1074-8, 1080. DOI: 10.2144/03345dd04. View

11.

Delzor A, Aurelie D, Escartin C, Carole E, Deglon N, Nicole D . Lentiviral vectors: a powerful tool to target astrocytes in vivo. Curr Drug Targets. 2013; 14(11):1336-46. DOI: 10.2174/13894501113146660213. View

12.

Matsuoka H, Miyake K, Shimada T . Improved methods of HIV vector mediated gene transfer. Int J Hematol. 1998; 67(3):267-73. DOI: 10.1016/s0925-5710(97)90114-6. View

13.

Schambach A, Baum C . Clinical application of lentiviral vectors - concepts and practice. Curr Gene Ther. 2008; 8(6):474-82. DOI: 10.2174/156652308786848049. View

14.

Kutner R, Zhang X, Reiser J . Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc. 2009; 4(4):495-505. DOI: 10.1038/nprot.2009.22. View

15.

Zhang C, Atasoy D, Arac D, Yang X, Fucillo M, Robison A . Neurexins physically and functionally interact with GABA(A) receptors. Neuron. 2010; 66(3):403-16. PMC: 3243752. DOI: 10.1016/j.neuron.2010.04.008. View

16.

Kohno T, Mohan S, Goto T, Morita C, Nakano T, Hong W . A new improved method for the concentration of HIV-1 infective particles. J Virol Methods. 2002; 106(2):167-73. DOI: 10.1016/s0166-0934(02)00162-3. View

17.

Teschemacher A, Wang S, Lonergan T, Duale H, Waki H, Paton J . Targeting specific neuronal populations using adeno- and lentiviral vectors: applications for imaging and studies of cell function. Exp Physiol. 2004; 90(1):61-9. DOI: 10.1113/expphysiol.2004.028191. View

18.

Tonini T, Claudio P, Giordano A, Romano G . Transient production of retroviral- and lentiviral-based vectors for the transduction of Mammalian cells. Methods Mol Biol. 2004; 285:141-8. DOI: 10.1385/1-59259-822-6:141. View

19.

Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage F . In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259):263-7. DOI: 10.1126/science.272.5259.263. View

20.

Papanikolaou E, Kontostathi G, Drakopoulou E, Georgomanoli M, Stamateris E, Vougas K . Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation. Virus Res. 2013; 175(1):1-11. DOI: 10.1016/j.virusres.2013.03.015. View