Inducible, tightly regulated and non-leaky neuronal gene expression in mice

Abstract

The Tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgene expression in mice with over 500 lines created to date. Although this system has been optimized over the years, it still has limitations such as residual transgene expression when turned off, referred to as leakiness. Here, we present a series of new Tet-OFF transgenic mice based on the second generation tetracycline-responsive transactivator system. The tTA-Advanced (tTA2S) is expressed under control of the neuron-specific Thy1.2 promoter (Thy-OFF), to regulate expression in the mouse brain. In addition, we generated a lacZ reporter line, utilizing the P tight Tet-responsive promoter (P tight–lacZ), to test our system. Two Thy-OFF transgenic lines displaying two distinct patterns of expression were selected. Oral doxycycline treatment of Thy-OFF/P tight–lacZ mice demonstrated tight transgene regulation with no leak expression. These new Thy-OFF mice are valuable for studies in a broad range of neurodegenerative diseases such as Alzheimer’s disease and related forms of dementia, where control of transgene expression is critical to understanding mechanisms underlying the disease. Furthermore, P tight–lacZ reporter mice may be widely applicable.

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References

  1. Baron U, Gossen M, Bujard H (1997) Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res 25(14):2723–2729

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  2. Blau HM, Rossi FM (1999) Tet B or not tet B: advances in tetracycline-inducible gene expression. Proc Natl Acad Sci USA 96(3):797–799

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  3. Chow JD, Price JT, Bills MM, Simpson ER, Boon WC (2012) A doxycycline-inducible, tissue-specific aromatase-expressing transgenic mouse. Transgenic Res 21(2):415–428. doi:10.1007/s11248-011-9525-7

    CAS  PubMed  Article  Google Scholar 

  4. Deuschle U, Meyer WK, Thiesen HJ (1995) Tetracycline-reversible silencing of eukaryotic promoters. Mol Cell Biol 15(4):1907–1914

    CAS  PubMed Central  PubMed  Google Scholar 

  5. Dewachter I, Reverse D, Caluwaerts N, Ris L, Kuiperi C, Van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, Van Leuven F (2002) Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci Off J Soc Neurosci 22(9):3445–3453. doi:20026290

    CAS  Google Scholar 

  6. Dobrovolsky VN, Heflich RH (2007) On the use of the T-REx tetracycline-inducible gene expression system in vivo. Biotechnol Bioeng 98(3):719–723. doi:10.1002/bit.21454

    CAS  PubMed  Article  Google Scholar 

  7. Duerr J, Gruner M, Schubert SC, Haberkorn U, Bujard H, Mall MA (2011) Use of a new-generation reverse tetracycline transactivator system for quantitative control of conditional gene expression in the murine lung. Am J Respir Cell Mol Biol 44(2):244–254. doi:10.1165/rcmb.2009-0115OC

    CAS  PubMed  Article  Google Scholar 

  8. Forster K, Helbl V, Lederer T, Urlinger S, Wittenburg N, Hillen W (1999) Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Nucleic Acids Res 27(2):708–710

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  9. Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H, Hennighausen L (1994) Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 91(20):9302–9306

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  10. Gilbert DM, Heery DM, Losson R, Chambon P, Lemoine Y (1993) Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1. Mol Cell Biol 13(1):462–472

    CAS  PubMed Central  PubMed  Google Scholar 

  11. Gill G, Ptashne M (1988) Negative effect of the transcriptional activator GAL4. Nature 334(6184):721–724. doi:10.1038/334721a0

    CAS  PubMed  Article  Google Scholar 

  12. Heinz N, Hennig K, Loew R (2013) Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator. BMC Biotechnol 13:5. doi:10.1186/1472-6750-13-5

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  13. Ittner LM, Gotz J (2007) Pronuclear injection for the production of transgenic mice. Nat Protoc 2(5):1206–1215. doi:10.1038/nprot.2007.145

    CAS  PubMed  Article  Google Scholar 

  14. Ittner LM, Fath T, Ke YD, Bi M, van Eersel J, Li KM, Gunning P, Gotz J (2008) Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia. Proc Natl Acad Sci USA 105(41):15997–16002. doi:10.1073/pnas.0808084105

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  15. Jones J, Nivitchanyong T, Giblin C, Ciccarone V, Judd D, Gorfien S, Krag SS, Betenbaugh MJ (2005) Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells. Biotechnol Bioeng 91(6):722–732. doi:10.1002/bit.20566

    CAS  PubMed  Article  Google Scholar 

  16. Kamper MR, Gohla G, Schluter G (2002) A novel positive tetracycline-dependent transactivator (rtTA) variant with reduced background activity and enhanced activation potential. FEBS Lett 517(1–3):115–120

    CAS  PubMed  Article  Google Scholar 

  17. Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H, Bujard H (1996) Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA 93(20):10933–10938

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  18. Rao P, Monks DA (2009) A tetracycline-inducible and skeletal muscle-specific Cre recombinase transgenic mouse. Dev Neurobiol 69(6):401–406. doi:10.1002/dneu.20714

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  19. Schonig K, Freundlieb S, Gossen M (2013) Tet-Transgenic Rodents: a comprehensive, up-to date database. Transgenic Res 22(2):251–254. doi:10.1007/s11248-012-9660-9

    PubMed Central  PubMed  Article  Google Scholar 

  20. Shaikh S, Nicholson LF (2006) Optimization of the Tet-On system for inducible expression of RAGE. J Biomol Tech 17(4):283–292

    PubMed Central  PubMed  Google Scholar 

  21. Sheng Y, Lin CC, Yue J, Sukhwani M, Shuttleworth JJ, Chu T, Orwig KE (2010) Generation and characterization of a Tet-On (rtTA-M2) transgenic rat. BMC Dev Biol 10:17. doi:10.1186/1471-213X-10-17

    PubMed Central  PubMed  Article  Google Scholar 

  22. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21(1):70–71. doi:10.1038/5007

    CAS  PubMed  Article  Google Scholar 

  23. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W (2000) Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 97(14):7963–7968. doi:10.1073/pnas.130192197

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  24. Valencik ML, McDonald JA (2001) Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart. Transgenic Res 10(3):269–275

    CAS  PubMed  Article  Google Scholar 

  25. Wells KD, Foster JA, Moore K, Pursel VG, Wall RJ (1999) Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch. Transgenic Res 8(5):371–381

    CAS  PubMed  Article  Google Scholar 

  26. Zhu Z, Ma B, Homer RJ, Zheng T, Elias JA (2001) Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice. J Biol Chem 276(27):25222–25229. doi:10.1074/jbc.M101512200

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

This work has been funded by the National Health & Medical Research Council (NH&MRC, Australia), the Australian Research Council (ARC), the Alzheimer Association (US), Alzheimer’s Australia, and the Jane Mason and Harold Stannett Williams Memorial Foundation (Australia). L.M.I. is an NH&MRC Senior Research Fellow. F.D. is supported by the Phillip Jacoby Scholarship in Alzheimer’s Dementia Research. J.M.W. is supported by the Rheumatic Disease Core Center.

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Correspondence to Lars M. Ittner.

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Supplementary Fig. 1 Expression patterns of the four different reporter lines generated. X-Gal staining of the brain of the four initial different reporter lines generated revealed most widespread staining for line #31. All lines have been crossed with the same inducer line (Thy-OFF15) for comparison (PDF 508 kb)

Supplementary Fig. 2 X-Gal staining of various organs reveals the specificity of the thy1.2 promoter. X-Gal staining of spinal cord, heart, liver and muscle of the wiltype (A), P tight–lacZ31 (B), Thy-OFF6 (C) and Thy-OFF 15 (C), did not reveal expression. In contrast, Thy-OFF6/P tight–lacZ31 and Thy-OFF15/P tight–lacZ31 mice exhibit strong β-galactosidas expression in the spinal cord only. Interestingly, Thy-cre/R26R-lacZ mice show some expression outside the CNS (PDF 862 kb)

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Delerue, F., White, M. & Ittner, L.M. Inducible, tightly regulated and non-leaky neuronal gene expression in mice. Transgenic Res 23, 225–233 (2014). https://doi.org/10.1007/s11248-013-9767-7

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Keywords

  • TET system
  • Transgenic mice
  • Doxycycline
  • LacZ reporter
  • Leakage
  • Thy1.2 promoter