Advertisement

Schwann Cells pp 177-193 | Cite as

Lentiviral Transduction of Rat Schwann Cells and Dorsal Root Ganglia Neurons for In Vitro Myelination Studies

  • Corey Heffernan
  • Patrice MaurelEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1739)

Abstract

Lentiviral transduction is a gene delivery method that provides numerous advantages over direct transfection and traditional retroviral or adenoviral delivery methods. It facilitates for the transduction of primary cells inherently difficult to transfect, delivers constructs of interest to nondividing as well as dividing cells, and permits the long-term expression of sizable DNA inserts (e.g., <7 kb). The study of peripheral nerve myelination at the molecular level has long benefited from the Schwann cells/dorsal root ganglia (DRG) neurons myelinating co-culture system. As this culture system takes about a month to develop and perform experiments with, lentiviral-delivered constructs can be used to manipulate gene expression in Schwann cells and DRG neurons, primary cells that are otherwise resilient to direct transfection. Here we present our protocol for lentiviral production and purification and subsequent infection of large numbers of Schwann cells and/or DRG neurons for the molecular study of peripheral nerve myelination in vitro.

Key words

Lentivirus Schwann cell Dorsal root ganglia neuron 

References

  1. 1.
    Ganem D, Nussbaum AL, Davoli D, Fareed GC (1976) Propagation of a segment of bacteriophage lambda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome. Cell 7(3):349–359CrossRefPubMedGoogle Scholar
  2. 2.
    Goff SP, Berg P (1976) Construction of hybrid viruses containing SV40 and lambda phage DNA segments and their propagation in cultured monkey cells. Cell 9(4 PT 2):695–705CrossRefPubMedGoogle Scholar
  3. 3.
    Nussbaum AL, Davoli D, Ganem D, Fareed GC (1976) Construction and propagation of a defective simian virus 40 genome bearing an operator from bacteriophage lambda. Proc Natl Acad Sci U S A 73(4):1068–1072CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mann R, Mulligan RC, Baltimore D (1983) Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33(1):153–159CrossRefPubMedGoogle Scholar
  5. 5.
    Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259):263–267CrossRefPubMedGoogle Scholar
  6. 6.
    Cronin J, Zhang XY, Reiser J (2005) Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther 5(4):387–398CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kaplan AH, Swanstrom R (1991) Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments. Proc Natl Acad Sci U S A 88(10):4528–4532CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nie Z, Phenix BN, Lum JJ, Alam A, Lynch DH, Beckett B, Krammer PH, Sekaly RP, Badley AD (2002) HIV-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation. Cell Death Differ 9(11):1172–1184. https://doi.org/10.1038/sj.cdd.4401094 CrossRefPubMedGoogle Scholar
  9. 9.
    Hoffmann M, Wu YJ, Gerber M, Berger-Rentsch M, Heimrich B, Schwemmle M, Zimmer G (2010) Fusion-active glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shut-off activity. J Gen Virol 91(Pt 11):2782–2793. https://doi.org/10.1099/vir.0.023978-0 CrossRefPubMedGoogle Scholar
  10. 10.
    Mitta B, Rimann M, Fussenegger M (2005) Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles. Metab Eng 7(5–6):426–436. https://doi.org/10.1016/j.ymben.2005.06.006 CrossRefPubMedGoogle Scholar
  11. 11.
    al Yacoub N, Romanowska M, Haritonova N, Foerster J (2007) Optimized production and concentration of lentiviral vectors containing large inserts. J Gene Med 9(7):579–584. https://doi.org/10.1002/jgm.1052
  12. 12.
    Kumar M, Keller B, Makalou N, Sutton RE (2001) Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther 12(15):1893–1905. https://doi.org/10.1089/104303401753153947 CrossRefPubMedGoogle Scholar
  13. 13.
    Chen Y, Ott CJ, Townsend K, Subbaiah P, Aiyar A, Miller WM (2009) Cholesterol supplementation during production increases the infectivity of retroviral and lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Biochem Eng J 44(2–3):199–207. https://doi.org/10.1016/j.bej.2008.12.004 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ciftci K, Levy RJ (2001) Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts. Int J Pharm 218(1–2):81–92CrossRefPubMedGoogle Scholar
  15. 15.
    Cribbs AP, Kennedy A, Gregory B, Brennan FM (2013) Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells. BMC Biotechnol 13:98. https://doi.org/10.1186/1472-6750-13-98 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Sakoda T, Kasahara N, Hamamori Y, Kedes L (1999) A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. J Mol Cell Cardiol 31(11):2037–2047. https://doi.org/10.1006/jmcc.1999.1035 CrossRefPubMedGoogle Scholar
  17. 17.
    Villa-Diaz LG, Garcia-Perez JL, Krebsbach PH (2010) Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix. Stem Cells Dev 19(12):1949–1957. https://doi.org/10.1089/scd.2009.0505 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Coelen RJ, Jose DG, May JT (1983) The effect of hexadimethrine bromide (polybrene) on the infection of the primate retroviruses SSV 1/SSAV 1 and BaEV. Arch Virol 75(4):307–311CrossRefPubMedGoogle Scholar
  19. 19.
    Manning JS, Hackett AJ, Darby NB Jr (1971) Effect of polycations on sensitivity of BALD-3T3 cells to murine leukemia and sarcoma virus infectivity. Appl Microbiol 22(6):1162–1163PubMedPubMedCentralGoogle Scholar
  20. 20.
    Toyoshima K, Vogt PK (1969) Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 38(3):414–426CrossRefPubMedGoogle Scholar
  21. 21.
    Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Zhang M, Ihrig MM, McManus MT, Gertler FB, Scott ML, Van Parijs L (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33(3):401–406. https://doi.org/10.1038/ng1117.ng1117[pii]CrossRefPubMedGoogle Scholar
  22. 22.
    Felgner PL (1990) Particulate systems and polymers for in vitro and in vivo delivery of polynucleotides. Adv Drug Deliv Rev 5(3):163–304CrossRefGoogle Scholar
  23. 23.
    Howell DP, Krieser RJ, Eastman A, Barry MA (2003) Deoxyribonuclease II is a lysosomal barrier to transfection. Mol Ther 8(6):957–963CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Biological SciencesRutgers UniversityNewarkUSA

Personalised recommendations