Virus Genes

, Volume 36, Issue 1, pp 55–66 | Cite as

Evaluation of the T-REx™ transcription switch for conditional expression and regulation of HSV-1 vectors

  • Charles Werner KnopfEmail author
  • Oksana Zavidij
  • Ingeborg Rezuchova
  • Július Rajčáni


Herpes simplex virus 1 (HSV-1) strain ANG and ANGpath were cloned as bacterial artificial chromosome (BAC). Two different types of BAC genomes were obtained. BAC genomes of type I contained the BAC replicon at the intended target region between the genes of UL48 and UL49. In BAC genomes of type II, the BAC sequences were found to be aberrantly fused between the termini of the HSV-1 genome. Both the BAC types were used to establish a conditional gene expression system for HSV-1 by Flp recombinase-mediated insertion of expression vectors that were modified to respond to the T-REx™ tetracycline (Tet)-inducible transcription switch. During BAC cloning and mutagenesis in E. coli, not only deletions but also defined mutations of the HSV-1 genome were observed. Successful virus reconstitution from BACs with large inserts demonstrated that HSV-1 has a packaging capacity for foreign sequences of at least 8.1% of its genome size. Targets for Tet-regulated gene expression were the viral DNA polymerase gene (pol) and a reporter gene of glycoprotein B fused to enhanced green fluorescent protein (gBGFP). Results with the pol gene as target showed that virus plaque production could not be significantly controlled by the T-REx™ gene switch using vectors encoding one copy of the tetR gene. In contrast, an efficient Tet-response was achieved with the gBGFP reporter, which was optimal in a Tet repressor (TetR)-producing cell line, demonstrating that the TetR concentration provided by the virus was not sufficient for a tight control of Tet-regulated gene expression.


HSV-1 ANG ANGpath BAC cloning Red/ET recombination Tetracycline T-REx™ transcription switch Gene expression 



This research was supported by a BMBF grant for the scientific and technical collaboration with the Slovakian Republic, Project SVK No. 00/006.


  1. 1.
    E.A. Burton, D.J. Fink, J. Glorioso, DNA Cell. Biol. 21, 915–936 (2002)PubMedCrossRefGoogle Scholar
  2. 2.
    E.-M. Borst, I. Crnkovic-Mertens, M. Messerle, in Methods in Molecular Biology, vol. 256: Bacterial Artificial Chromosomes, vol. 2: Functional Studies, eds. by S. Zhao, M. Stodolsky (Humana Press Inc., Totowa, 2004), pp. 221–239Google Scholar
  3. 3.
    G.A. Smith, L.W. Enquist, Proc Natl. Acad. Sci. USA 97, 4873–4878 (2000)PubMedCrossRefGoogle Scholar
  4. 4.
    R. Rupp, Z. Ruzsics, T. Sacher, U.H. Koszinowski, J. Virol. 79, 486–494 (2005)PubMedCrossRefGoogle Scholar
  5. 5.
    F. Yao, T. Svensjo, T. Winkler, M. Lu, C. Eriksson, E. Eriksson, Hum. Gene Ther. 9, 1939–1950 (1998)PubMedGoogle Scholar
  6. 6.
    P.P. Cherepanov, W. Wackernagel, Gene 158, 9–14 (1995)PubMedCrossRefGoogle Scholar
  7. 7.
    F. Yao, C. Theopold, D. Hoeller, O. Bleiziffer, Z. Lu, Mol. Ther. 13, 1133–1141 (2006)PubMedCrossRefGoogle Scholar
  8. 8.
    Y.F. Zhang, F. Buchholz, J.P. Muyrers, A.F. Stewart, Nat. Genet. 20, 123–128 (1998)PubMedCrossRefGoogle Scholar
  9. 9.
    R. Strick, J. Hansen, R. Bracht, D. Komitowski, C.W. Knopf, Intervirology 40, 41–49 (1997)PubMedGoogle Scholar
  10. 10.
    D.J. McGeoch, M.A. Dalrymple, A.J. Davison, A. Dolan, M.C. Frame, D. McNab, L.J. Perry, J.E. Scott, P. Taylor, J. Gen. Virol. 69, 1531–1574 (1988)PubMedGoogle Scholar
  11. 11.
    K.A. Datsenko, B.L. Wanner, Proc Natl. Acad. Sci. USA 97, 66640–6645 (2000)CrossRefGoogle Scholar
  12. 12.
    R. Atalay, A. Zimmermann, M. Wagner, E. Borst, C. Benz, M. Messerle, H. Hengel, J. Virol. 76, 8596–8608 (2002)PubMedCrossRefGoogle Scholar
  13. 13.
    H. Adler, M. Messerle, M. Wagner, U.H. Koszinowski, J. Virol. 74, 6964–6974 (2000)PubMedCrossRefGoogle Scholar
  14. 14.
    Borst E.M., M. Messerle, J. Virol. 79, 3615–3626 (2005)PubMedCrossRefGoogle Scholar
  15. 15.
    R. Kolter, M. Inuzuka, D.P. Helinski, Cell 15, 1199–1208 (1978)PubMedCrossRefGoogle Scholar
  16. 16.
    M. Kozak, Nucl. Acids Res. 15, 8125–8148 (1987)PubMedCrossRefGoogle Scholar
  17. 17.
    F.J.P. Kühn, C.W. Knopf, J. Biol. Chem. 271, 29245–29254 (1996)PubMedCrossRefGoogle Scholar
  18. 18.
    B. Hirt, J. Mol. Biol. 26, 365–369 (1967)PubMedCrossRefGoogle Scholar
  19. 19.
    M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Zeigler, U.H. Koszinowski, Proc. Natl. Acad. Sci. USA 94, 14759–14763 (1997)PubMedCrossRefGoogle Scholar
  20. 20.
    J.F. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000)Google Scholar
  21. 21.
    C.P. Gray, H.C. Kaerner, J. Gen. Virol. 65, 2109–2119 (1984)PubMedCrossRefGoogle Scholar
  22. 22.
    J. Kosovsky, A. Vojvodova, I. Oravcova, M. Kudelova, J. Matis, J. Rajčani, Virus Genes 20, 27–33 (2000)PubMedCrossRefGoogle Scholar
  23. 23.
    E.-M. Borst, G. Hahn, U.H. Koszinowski, M. Messerle, J. Virol. 73, 8320–8329 (1999)PubMedGoogle Scholar
  24. 24.
    R. Wade-Martins, E.R. Smith, E. Tyminski, E.A. Chiocca, Y. Saeki, Nat. Biotech. 19, 1067–1070 (2001)CrossRefGoogle Scholar
  25. 25.
    J.P. Quinn, D.J. McGeoch, Nucl. Acids Res. 13, 8143–8163 (1985)PubMedCrossRefGoogle Scholar
  26. 26.
    R.R. Spaete, N. Frenkel, Proc. Natl. Acad. Sci. USA 82, 694–698 (1985)PubMedCrossRefGoogle Scholar
  27. 27.
    C.W. Knopf, B. Spies, H.C. Kaerner, Nucl. Acids Res. 14, 8655–8667 (1986)PubMedCrossRefGoogle Scholar
  28. 28.
    S.K. Weller, A. Spadaro, J.E. Schaffer, A.W. Murray, A.M. Maxam, P.A. Schaffer, Mol. Cell. Biol. 5, 930–942 (1985)PubMedGoogle Scholar
  29. 29.
    D. Lockshon, D.A. Galloway, J. Virol. 58, 513–521 (1986)PubMedGoogle Scholar
  30. 30.
    M.A. Hardwicke, P.A. Schaffer, J. Virol. 69, 1377–1388 (1995)PubMedGoogle Scholar
  31. 31.
    J.W. Balliet, P.A. Schaffer, J. Virol. 80, 440–450 (2006)PubMedCrossRefGoogle Scholar
  32. 32.
    J. Song, F. Dong, J.W. Lilly, R.M. Stupar, J. Jiang, Genome 44, 463–469 (2001)PubMedCrossRefGoogle Scholar
  33. 33.
    S. Freundlieb, C. Schirra-Müller, H. Bujard, J. Gen. Med. 1, 4–12 (1999)CrossRefGoogle Scholar
  34. 34.
    G.H. Patterson, S.M. Knobel, W.D. Sharif, S.R. Kain, D.W. Piston, Biophys. J. 73, 2782–2790 (1997)PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Charles Werner Knopf
    • 1
    Email author
  • Oksana Zavidij
    • 1
  • Ingeborg Rezuchova
    • 2
  • Július Rajčáni
    • 2
  1. 1.Forschungsschwerpunkt Angewandte TumorvirologieDeutsches Krebsforschungszentrum, DKFZ - ATVHeidelbergGermany
  2. 2.Institute of VirologySASBratislavaSlovakia

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