Molecular Biotechnology

, Volume 59, Issue 6, pp 200–206 | Cite as

Design of a Lentiviral Vector for the Inducible Expression of MYC: A New Strategy for Construction Approach

  • Onur Tokgun
  • Francesco Paolo Fiorentino
  • Pervin Elvan Tokgun
  • Jun Yokota
  • Hakan Akca
Original Paper


Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.


Lentivirus Myc MCS SCLC Dox Inducible vector 



Onur Tokgun acknowledges the support from TUBITAK (International Research Fellowship Program 2214/A). F.P. Fiorentino acknowledges the support from Fondazione Umberto Veronesi. This study was supported by Pamukkale University Scientific Research Projects Coordination Unit (2013SBE012 and 2016HZDP007).

Compliance with Ethical Standards

Conflict of interest

Authors declared that there is no competing interest.


  1. 1.
    Vargas, J. E., Chicaybam, L., Stein, R. T., Tanuri, A., Delgado-Cañedo, A., & Bonamino, M. H. (2016). Retroviral vectors and transposons for stable gene therapy: Advances, current challenges and perspectives. Journal of Translational Medicine, 14, 288.CrossRefGoogle Scholar
  2. 2.
    Howarth, J. L., Lee, Y. B., & Uney, J. B. (2010). Using viral vectors as gene transfer tools. Cell Biology and Toxicology, 26(1), 1–20.CrossRefGoogle Scholar
  3. 3.
    Oleg, T. (2009). Designing plasmid vectors. In Walther, W., Stein, U.S. (Eds.), Gene therapy of cancer, vol.542: Methods in molecular biology (pp. 117–129). Totowa, NJ: Humana.Google Scholar
  4. 4.
    Tan, J. Y., Sellers, D. L., Pham, B., Pun, S. H., & Horner, P. J. (2016). Non-viral nucleic acid delivery strategies to the central nervous system. Frontiers in Molecular Neuroscience, 9, 108.CrossRefGoogle Scholar
  5. 5.
    Schleef, M., Blaesen, M., Schmeer, M., Baier, R., Marie, C., Dickson, G., et al. (2010). Production of non viral DNA vectors. Current Gene Therapy, 10(6), 487–507.CrossRefGoogle Scholar
  6. 6.
    Ni, R., Zhou, J., Hossain, N., & Chau, Y. (2016). Virus-inspired nucleic acid delivery system: Linking virus and viral mimicry. Advanced Drug Delivery Reviews, 106(Pt A), 3–26.CrossRefGoogle Scholar
  7. 7.
    Bouard, D., Alazard-Dany, N., & Cosset, F.-L. (2009). Viral vectors: From virology to transgene expression. British Journal of Pharmacology, 157(2), 153–165.CrossRefGoogle Scholar
  8. 8.
    Kay, M. A., Glorioso, J. C., & Naldini, L. (2001). Viral vectors for gene therapy: The art of turning infectious agents into vehicles of therapeutics. Nature Medicine, 7, 33–40.CrossRefGoogle Scholar
  9. 9.
    Jung, P., Menssen, A., Mayr, D., & Hermeking, H. (2008). AP4 encodes a c-MYC-inducible repressor of p21. PNAS, 105(39), 15046–15051.CrossRefGoogle Scholar
  10. 10.
    Yamamizu, K., Sharov, A. A., Piao, Y., Amano, M., Yu, H., Nishiyama, A., et al. (2016). Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines. Scientific Reports, 6, 25667.CrossRefGoogle Scholar
  11. 11.
    Herdegen, T., & Leah, J. D. (1998). Inducible and constitutive transcription factors in the mammalian nervous system: Control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Research. Brain Research Reviews, 28(3), 370–490.CrossRefGoogle Scholar
  12. 12.
    Moumtzi, S. S., Roberts, M. L., Joyce, T., Evangelidou, M., Probert, L., Frillingos, S., et al. (2010). Gene expression profile associated with oncogenic ras-induced senescence, cell death, and transforming properties in human cells. Cancer Investigation, 28(6), 563–587.CrossRefGoogle Scholar
  13. 13.
    Schmetsdorf, S., Gärtner, U., & Arendt, T. (2007). Constitutive expression of functionally active cyclin-dependent kinases and their binding partners suggests noncanonical functions of cell cycle regulators in differentiated neurons. Cerebral Cortex, 17(8), 1821–1829.CrossRefGoogle Scholar
  14. 14.
    Dauphinot, L., De Oliveira, C., Melot, T., Sevenet, N., Thomas, V., Weissman, B. E., et al. (2001). Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2and c-Myc expression. Oncogene, 20(25), 3258–3265.CrossRefGoogle Scholar
  15. 15.
    Matsushita, N., Matsushita, S., Hirakawa, S., & Higashiyama, S. (2013). Doxycycline-dependent inducible and reversible RNA interference mediated by a single lentivirus vector. Bioscience, Biotechnology, and Biochemistry, 77(4), 776–781.CrossRefGoogle Scholar
  16. 16.
    Mullick, A., Xu, Y., Warren, R., Koutroumanis, M., Guilbault, C., Broussau, S., et al. (2006). The cumate gene-switch: A system for regulated expression in mammalian cells. BMC Biotechnology, 6, 43.CrossRefGoogle Scholar
  17. 17.
    Durand, S., & Cimarelli, A. (2011). The inside out of lentiviral vectors. Viruses, 3(2), 132–159.CrossRefGoogle Scholar
  18. 18.
    Buchschacher, G. L., Jr., & Wong-Staal, F. (2000). Development of lentiviral vectors for gene therapy for human diseases. Blood, 95(8), 2499–2504.Google Scholar
  19. 19.
    Kotterman, M. A., Chalberg, T. W., & Schaffer, D. V. (2015). Viral vectors for gene therapy: Translational and clinical outlook. Annual Review of Biomedical Engineering, 17, 63–89.CrossRefGoogle Scholar
  20. 20.
    Jakobsson, J., & Lundberg, C. (2006). Lentiviral vectors for use in the central nervous system. Molecular Therapy, 13(3), 484–493.CrossRefGoogle Scholar
  21. 21.
    Sakemura, R., Terakura, S., Watanabe, K., Julamanee, J., Takagi, E., Miyao, K., et al. (2016). A Tet-On inducible system for controlling CD19-chimeric antigen receptor expression upon drug administration. Cancer Immunology Research, 4(8), 658–668.CrossRefGoogle Scholar
  22. 22.
    Bai, J., Li, J., & Mao, Q. (2013). Construction of a single lentiviral vector containing tetracycline-ınducible Alb-uPA for transduction of uPA expression in murine hepatocytes. PLoS ONE, 8(4), e61412.CrossRefGoogle Scholar
  23. 23.
    Johansen, J., Rosenblad, C., Andsberg, K., Moller, A., Lundberg, C., Bjorlund, A., et al. (2002). Evaluation of Tet-on system to avoid transgene downregulation in ex vivo gene transfer to the CNS. Gene Therapy, 9, 1291–1301.CrossRefGoogle Scholar
  24. 24.
    Papadakis, E. D., Nicklin, S. A., Baker, A. H., & White, S. J. (2004). Promoters and control elements: Designing expression cassettes for gene therapy. Current Gene Therapy, 4(1), 89–113.CrossRefGoogle Scholar
  25. 25.
    Zhou, X., Vink, M., Klaver, B., Berkhout, B., & Das, A. T. (2006). Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Therapy, 13(19), 1382–1390.CrossRefGoogle Scholar
  26. 26.
    Ciuffi, A. (2008). Mechanisms governing lentivirus integration site selection. Current Gene Therapy, 8(6), 419–429.CrossRefGoogle Scholar
  27. 27.
    Kappel, S., Matthess, Y., Kaufmann, M., & Strebhardt, K. (2007). Silencing of mammalian genes by tetracycline-inducible shRNA expression. Nature Protocols, 2(12), 3257–3269.CrossRefGoogle Scholar
  28. 28.
    Krueger, C., Pfleiderer, K., Hillen, W., & Berens, C. (2004). Tetracycline derivatives: Alternative effectors for Tet transregulators. Biotechniques, 37(4), 546, 548, 550.Google Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Onur Tokgun
    • 1
  • Francesco Paolo Fiorentino
    • 2
    • 3
  • Pervin Elvan Tokgun
    • 1
  • Jun Yokota
    • 4
  • Hakan Akca
    • 1
  1. 1.Department of Medical BiologyPamukkale University School of MedicineDenizliTurkey
  2. 2.Kitos Biotech srls, Porto Conte RicercheAlgheroItaly
  3. 3.Department of Biomedical SciencesUniversity of SassariSassariItaly
  4. 4.Genomics and Epigenomics of Cancer Prediction ProgramInstitut d’Investigació Germans Trias i Pujol (IGTP)BarcelonaSpain

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