Transgenic Research

, Volume 12, Issue 1, pp 33–43 | Cite as

Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

  • Mischala A. Grill
  • Mark A. Bales
  • Amber N. Fought
  • Kristopher C. Rosburg
  • Stephanie J. Munger
  • Parker B. Antin


Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

doxycycline muscle creatine kinase skeletal muscle tet-on transgenic mice 


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  1. Baron U and Bujard H (2000) Tet repressor-based system for regulated gene expression in eukaryotic cells: Principles and advances. Method Enzymol 327: 401–421.Google Scholar
  2. Busch WA, Stromer MH, Goll DE and Suzuki A (1972) Cal2+ specific removal of Z-lines from rabbit skeletal muscle. J Cell Biol 52: 367–381.Google Scholar
  3. Cameron-Smith D (2002) Exercise and skeletal muscle gene expression. Clin Exp Pharmacol P 29: 209–213.Google Scholar
  4. Capecchi MR (2000) Choose your target. Nat Genet 26: 159–161.Google Scholar
  5. Cong M, Thompson VF, Goll DE and Antin PB (1998) The bovine calpastatin gene promoter and a new N-terminal region of the protein are targets for cAMP dependent protein kinase activity. J Biol Chem 273: 660–666.Google Scholar
  6. Cordier L, Hack AA, Scott MO, Barton-Davis ER, Gao G, Wilson JM, McNalley EM and Sweeney HL (2000) Rescue of skeletal mucles of gamma-sarcogylcan-deficient mice with adeno-associated virus-mediated gene transfer. Mol Ther 1: 119–129.Google Scholar
  7. Croall DE and Demartino GN (1991) Calcium-activated neutral protease (calpain) system: Structure, function and regulation. Biological Rev 71: 813–847.Google Scholar
  8. Dayton WR, Goll DE, Stromer MH, Reville WJ, Zeece MG and Robson RM (1975) Some properties of a calcium activated protease that may be involved in myofibrillar protein turnover. In: Reich E, Rifkin DB and Shaw E (eds), Cold Spring Harbor Conferences on Cell Proliferation. Vol. 2 (pp. 551–577) Cold Spring Harbor Press, Cold Spring Harbor, NY, USA.Google Scholar
  9. Delort JP and Capecchi MR (1996) TAXI/UAS: A molecular switch to control expression of genes in vivo. Hum Gene Ther 7: 809–820.Google Scholar
  10. Dobie K, Mehtali M, McClenaghan M and Lathe R (1997) Variegated gene expression in mice. Trends Genet 13: 127–130.Google Scholar
  11. Donoviel DB, Sheild MA, Buskin JN, Haugen HS, Clegg CH and Hauschka SD (1996) Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice. Mol Cell Biol 16: 1649–1658.Google Scholar
  12. Efrat S, Fusco-DeMane D, Lemberg H, al Emran O and Wang X (1995) Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Proc Natl Acad Sci USA 92: 3576–3580.Google Scholar
  13. Federov LM, Tyrsin OY, Krenn V, Chernigovskaya EV and Rapp UR (2001) Tet-system for the regulation of gene expression during embryonic development. Transgenic Res 10: 247–258.Google Scholar
  14. Festenstein R, Tolaini M, Corbella P, Mamalaki C, Parrington J, Fox M, Miliou A, Jones M and Kioussis D (1996) Locus control region fucntion and hetrochromatin-induced position effect variegation. Science 271: 1123–1125.Google Scholar
  15. Freundlieb S, Schirra-Muller C and Bujard H (1999) A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med 1: 4–12.Google Scholar
  16. Furth PA, St Onge L, Böger G, Gruss P, Gossen M, Kistner A, Bujard H and Hennighausen L (1994) Temporal control of gene expression in transgenic mice by a tetracylcine-responsive promoter. Proc Natl Acad Sci USA 91: 9302–9306.Google Scholar
  17. Gao X, Kemper A and Popko B (1999) Advanced transgenic and gene-targeting approaches. Neurochem Res 24: 1181–1188.Google Scholar
  18. Garrick D, Fiering S, Martin DIK and Whitelaw E (1998) Repeatinduced gene silencing in mammals. Nat Genet 18: 56–59.Google Scholar
  19. Goll DE (2002) The calpain system in muscle tissue (in press).Google Scholar
  20. Gossen M and Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547–5551.Google Scholar
  21. Grosveld F, van Assendelft GB, Greaves DR and Kollias G (1987) Postion-independent, high level expression of the human b-globin gene in transgenic mice. Cell 51: 975–985.Google Scholar
  22. Guy LG, Kothary R and Wall L (1997) Position effects in mice carrying LacZ transgene in cis with the beta-globin LCR can be explained by a graded model. Nucleic Acids Res 25: 4400–4407.Google Scholar
  23. Johnson JE, Wold BJ and Hauschka SD (1989) Muscle creatine kinase sequence elements regulating skeletal and cardiac expression in transgenic mice. Mol Cell Biol 9: 3393–3399.Google Scholar
  24. Kistner A, Gossen M, Zimmerman F, Jerecic J, Ullmer C, Lübbert H and Bujard H (1998) Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA 93: 10933–10938.Google Scholar
  25. Lamartina S, Roscilli G, Rinaudo CD, Sporeno E, Silvi L, Hillen W, Bujard H, Cortese R, Ciliberto G and Toniatti C (2002) Stringent control of gene expression in vivo by using novel doxycyclinedependent trans-activators. Hum Gene Ther 13: 199–210.Google Scholar
  26. Larochelle N, Qualikene W, Dunant P, Massie B, Karpati G, Nalbantoglu J and Lochmuller H (2002) The short MCK1350 promoter/enhancer allows for sufficient dystrophin expression in skeletal muscles of transgenic mice. Biochem Bioph Res Comm 292: 626–631.Google Scholar
  27. Lewandoski M (2001) Conditional control of gene expresion in the mouse. Nat Rev Genet 2 Google Scholar
  28. Martin DI and Whitelaw E (1996) The vagaries of variegating transgenes. Bioessays 18: 919–923.Google Scholar
  29. Naya FJ, Mercer B, Shelton J, Richardson JA, Wiliams RS and Olson EN (2000) Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo. J Biol Chem 275: 4545–4548.Google Scholar
  30. No D, Yao TP and Evans RM (1996) Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 93: 3346–3351.Google Scholar
  31. Opsahl ML, McClenaghan M, Springbett A, Reid S, Lathe R, Colman A and Whitelaw CBA (2002) Multiple effects of genetic background on variegated transgene expression inmice. Genetics 160: 1107–1112.Google Scholar
  32. Perl A-KT, Tichelaar JW and Whitsett JA (2002) Conditional gene expression in the respiratory epithelium of the mouse. Transgenic Res 11: 212–229.Google Scholar
  33. Robertson A, Perea J, Tolmachova R, Thomas PK and Huxley C (2002) Effects of mouse strain, position of integration and tetracycline analogue on the tetracycline conditional system in transgenic mice. Gene 282: 65–74.Google Scholar
  34. Sauer B (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14: 381–392.Google Scholar
  35. Sawicki JA, Monks B and Morris RJ (1998) Cell-specific ecdysoneinducible expression of FLP recombinase in mammalian cells. BioTechniques 25: 868–875.Google Scholar
  36. Schumacher A, Koetsier PA, Hertz J and Doerfler W (2000) Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice. J Biol Chem 275: 37915–37921.Google Scholar
  37. Shield MA, Haugen HS, Clegg CH and Hauschka SD (1996) Ebox sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice. Mol Cell Biol 16: 5058–5068.Google Scholar
  38. Shin MK, Levorse JM, Ingram RS and Tilghman SM (1999) The temporal requirement for endothelin receptor-B signaling during neural crest development. Nature 402: 496–501.Google Scholar
  39. Ueda Y, Wang M-C, Ou BR, Huang J, Elce J, Tanaka K, Ichihara A and Forsbert NE (1998) Evidence for the participation of the proteosome and calpain in early phases of muscle cell differentiation. Int J Biochem Cell B 30: 679–694.Google Scholar
  40. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H and Hillen W (2000) Exploring the sequence space for tetracyclinedependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 97: 7963–7968.Google Scholar
  41. Valencik ML and McDonald JA (2000) Codon optimization markedly improves doxycycline regulated expression in the mouse heart. Transgenic Res 10: 269–275.Google Scholar
  42. Yamamoto A, RH and Dauer WT (2001) The ons and offs of inducible transgenic technology: A review. Neurobiol Dis 8: 923–932.Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Mischala A. Grill
    • 1
  • Mark A. Bales
    • 1
  • Amber N. Fought
    • 1
  • Kristopher C. Rosburg
    • 1
  • Stephanie J. Munger
    • 1
  • Parker B. Antin
    • 1
  1. 1.Department of Cell Biology and AnatomyTucsonUSA

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