Transgenic Research

, Volume 17, Issue 2, pp 265–279 | Cite as

Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters

  • Christopher T. Burket
  • Jacob E. Montgomery
  • Ryan Thummel
  • Sean C. Kassen
  • Matthew C. LaFave
  • David M. Langenau
  • Leonard I. Zon
  • David R. HydeEmail author
Original Paper


Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.


Zebrafish Central nervous system Ubiquitous Promoter Tol2 Adult expression pattern 



Enhanced green fluorescent protein


Reverse transcriptase polymerase chain reaction


Polymerase chain reaction


Hours post-fertilization


Days post-fertilization



The authors thank the Freimann Life Science Center Staff for the care and maintenance of the zebrafish facility and Suzyanne Guzicki for the injection of the expression constructs into zebrafish embryos. We also thank Koichi Kawakami for the generous gift of the pT2KXIG construct containing the Tol2 transposable element and Tol2 transposase gene (pCSTZ2.8) and the Campos-Ortega lab for the gift of the H2A.F/Z promoter. This work was supported by the National Institute of Health (R21-EY017134 to D.R.H).


  1. Amsterdam A, Lin S, Hopkins N (1995) The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev Biol 171(1):123–129PubMedCrossRefGoogle Scholar
  2. Amsterdam A, Lin S, Moss LG, Hopkins N (1996) Requirements for green fluorescent protein detection in transgenic zebrafish embryos. Gene 173:99–103PubMedCrossRefGoogle Scholar
  3. Bai S, Thummel R, Godwin AR, Nagase H, Itoh Y, Li L, Evans R, McDermott J, Seiki M, Sarras MP Jr (2005) Matrix metalloproteinase expression and function during fin regeneration in zebrafish: analysis of MT1-MMP, MMP2 and TIMP2. Matrix Biol 24(4):247–260PubMedCrossRefGoogle Scholar
  4. Bretaud S, Li Q, Lockwood BL, Kobayashi K, Lin E, Guo S (2007) A choice behavior for morphine reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish. J Neurosci 146(3):1109–1116CrossRefGoogle Scholar
  5. Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD (1988) Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 85(3):836–840PubMedCrossRefGoogle Scholar
  6. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805PubMedCrossRefGoogle Scholar
  7. Collas P (1998) Modulation of plasmid DNA methylation and expression in zebrafish embryos. Nuc Acids Res 26(19):4454–4461CrossRefGoogle Scholar
  8. Dong J, Stuart GW (2004) Transgene manipulation in zebrafish by using recombinases. Methods Cell Biol 77:363–379PubMedGoogle Scholar
  9. Feng H, Langenau DM, Madge JA, Quinkertz A, Gutierrez A, Neuberg DS, Kanki JP, Look TA (2007) Heat-shock induction of T-cell lymphoma/leukaemia in conditional Cre/lox-regulated transgenic zebrafish. Br J Haematol 138(2):169–175PubMedCrossRefGoogle Scholar
  10. Fimbel SM, Montgomery JE, Burket CT, Hyde DR (2007) Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish. J Neurosci 27(7):1712–1724PubMedCrossRefGoogle Scholar
  11. Furutani-Seiki M, Jiang YJ, Brand M, Heisenberg CP, Houart C, Beuchle D, van Eeden FJ, Granato M, Haffter P, Hammerschmidt M, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C (1996) Neural degeneration mutants in the zebrafish, Danio rerio. Development 123:229–239PubMedGoogle Scholar
  12. Gerhard GS (2007) Small laboratory fish as models for aging research. Ageing Res Rev 6(1):64–72PubMedCrossRefGoogle Scholar
  13. Gibbs PD, Schmale MC (2000) GFP as a genetic marker scorable throughout the life cycle of transgenic zebra fish. Mar Biotechnol (NY) 2(2):107–125Google Scholar
  14. Gibbs PD, Peek A, Thorgaard G (1994) An in vivo screen for the luciferase transgene in zebrafish. Mol Mar Biol Biotechnol 3(6):307–316PubMedGoogle Scholar
  15. Grunwald DJ, Kimmel CB, Westerfield M, Walker C, Streisinger G (1988) A neural degeneration mutation that spares primary neurons in the zebrafish. Dev Biol 126(1):115–128PubMedCrossRefGoogle Scholar
  16. Herrera PL (2002) Defining the cell lineages of the islets of Langerhans using transgenic mice. Int J Dev Biol 46(1):97–103PubMedGoogle Scholar
  17. Herrera PL, Nepote V, Delacour A (2002) Pancreatic cell lineage analyses in mice. Endocrine 19(3):267–278PubMedCrossRefGoogle Scholar
  18. Herrera PL, Orci L, Vassalli JD (1998) Two transgenic approaches to define the cell lineages in endocrine pancreas development. Mol Cell Endocrinol 140(1–2):45–50PubMedCrossRefGoogle Scholar
  19. Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G (1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol 192(2):289–299PubMedCrossRefGoogle Scholar
  20. Hirsch N, Zimmerman LB, Gray J, Chae J, Curran KL, Fisher M, Ogino H, Grainger RM (2002) Xenopus tropicalis transgenic lines and their use in the study of embryonic induction. Dev Dyn 225(4):522–535PubMedCrossRefGoogle Scholar
  21. Iovine MK, Higgins EP, Hindes A, Coblitz B, Johnson SL (2005) Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins. Dev Biol 278(1):208–219PubMedCrossRefGoogle Scholar
  22. Johnson AD, Krieg PA (1994) pXeX, a vector for efficient expression of cloned sequences in Xenopus embryos. Gene 147(2):223–226PubMedCrossRefGoogle Scholar
  23. Johnson AD, Krieg PA (1995). A Xenopus laevis gene encoding EF-1 alpha S, the somatic form of elongation factor 1 alpha: sequence, structure, and identification of regulatory elements required for embryonic transcription. Dev Genet 17(3):280–290PubMedCrossRefGoogle Scholar
  24. Ju B, Xu Y, He J, Liao J, Yan T, Hew CL, Lam TJ, Gong Z (1999) Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters. Dev Genet 25(2):158–167PubMedCrossRefGoogle Scholar
  25. Kassen SC, Ramanan V, Montgomery JE, Burket CT, Liu CG, Vihtelic TS, Hyde DR (2007) Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish. Dev Neurobiol 67:1009–1031PubMedCrossRefGoogle Scholar
  26. Kawakami K, Koga A, Hori H, Shima A (1998) Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225(1–2):17–22PubMedCrossRefGoogle Scholar
  27. Kawakami K, Shima A (1999) Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240(1):239–244PubMedCrossRefGoogle Scholar
  28. Kawakami K, Shima A, Kawakami N (2000) Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci USA 97(21):11403–11408PubMedCrossRefGoogle Scholar
  29. Kennedy BN, Vihtelic TS, Checkley L, Vaughan KT, Hyde DR (2001) Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors. J Biol Chem 276(17):14037–14043PubMedGoogle Scholar
  30. Kim KH, Antkiewicz DS, Yan L, Eliceir KW, Heideman W, Peterson RE, Lee Y (2007) Lrrc10 is required for early heart development and function in zebrafish. Dev Biol 308(2):494–506PubMedCrossRefGoogle Scholar
  31. Kondo M (2007) Bone morphogenetic proteins in the early development of zebrafish. Febs J 274(12):2960–2967PubMedCrossRefGoogle Scholar
  32. Langenau DM, Feng H, Berghmans S, Kanki JP, Kutok JL, Look AT (2005) Cre/lox-regulated transgenic zebrafish model with conditional myc-induced T cell acute lymphoblastic leukemia. Proc Natl Acad Sci USA 102(17):6068–6073PubMedCrossRefGoogle Scholar
  33. Le Hir H, Nott A, Moore MJ (2003) How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci 28(4):215–220PubMedCrossRefGoogle Scholar
  34. Lee Y, Grill S, Sanchez A, Murphy-Ryan M, Poss KD (2005) Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration. Development 132(23):5173–5183PubMedCrossRefGoogle Scholar
  35. Lesaffre B, Joliot A, Prochiantz A, Volovitch M (2007) Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish. Neural Develop 2:2PubMedCrossRefGoogle Scholar
  36. Lewis KE, Eisen JS (2003) From cells to circuits: development of the zebrafish spinal cord. Prog Neurobiol 69(6):419–449PubMedCrossRefGoogle Scholar
  37. Long Q, Meng A, Wang H, Jessen JR, Farrell MJ, Lin S (1997) GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124(20):4105–4111PubMedGoogle Scholar
  38. Malicki J, Neuhauss SC, Schier AF, Solnica-Krezel L, Stemple DL, Stainier DY, Abdelilah S, Zwartkruis F, Rangini Z, Driever W (1996a) Mutations affecting development of the zebrafish retina. Development 123:263–273PubMedGoogle Scholar
  39. Malicki J, Schier AF, Solnica-Krezel L, Stemple DL, Neuhauss SC, Stainier DY, Abdelilah S, Rangini Z, Zwartkruis F, Driever W (1996b) Mutations affecting development of the zebrafish ear. Development 123:275–283PubMedGoogle Scholar
  40. Meng A, Tang H, Ong BA, Farrell MJ, Lin S (1997) Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2. Proc Natl Acad Sci USA 94(12):6267–6272PubMedCrossRefGoogle Scholar
  41. Muto A, Orger MB, Wehman AM, Smear MC, Kay JN, Page-McCaw PS, Gahtan E, Xiao T, Nevin LM, Gosse NJ, Staub W, Finger-Baier K, Baier H (2005) Forward genetic analysis of visual behavior in zebrafish. PLoS Genet 1(5):e66PubMedCrossRefGoogle Scholar
  42. Nakatani Y, Kawakami A, Kudo A (2007) Cellular and molecular processes of regeneration, with special emphasis on fish fins. Dev Growth Differ 49(2):145–154PubMedCrossRefGoogle Scholar
  43. Ninkovic J, Bally-Cuif L (2006) The zebrafish as a model system for assessing the reinforcing properties of drugs of abuse. Methods 39(3):262–274PubMedCrossRefGoogle Scholar
  44. Orger MB, Gahtan E, Muto A, Page-McCaw P, Smear MC, Baier H (2004) Behavioral screening assays in zebrafish. Methods Cell Biol 77:53–68PubMedGoogle Scholar
  45. Pan X, Wan H, Chia W, Tong Y, Gong Z (2005) Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system. Transgen Res 14(2):217–223CrossRefGoogle Scholar
  46. Pauls S, Geldmacher-Voss B, Campos-Ortega JA (2001) A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development. Dev Genes Evol 211(12):603–610PubMedCrossRefGoogle Scholar
  47. Poss KD, Wilson LG, Keating MT (2002) Heart regeneration in zebrafish. Science 298(5601):2188–2190PubMedCrossRefGoogle Scholar
  48. Schmitt EA, Dowling JE (1994) Early eye morphogenesis in the zebrafish, Brachydanio rerio. J Comp Neurol 344(4):532–542PubMedCrossRefGoogle Scholar
  49. Schmitt EA, Dowling JE (1999) Early retinal development in the zebrafish, Danio rerio: light and electron microscopic analyses. J Comp Neurol 404(4):515–536PubMedCrossRefGoogle Scholar
  50. Thummel R, Bai S, Sarras MP Jr, Song P, McDermott J, Brewer J, Perry M, Zhang X, Hyde DR, Godwin AR (2006a) Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb. Dev Dyn 235(2):336–346PubMedCrossRefGoogle Scholar
  51. Thummel R, Burket CT, Brewer JL, Sarras MP Jr., Li L, Perry M, McDermott JP, Sauer B, Hyde DR, Godwin AR (2005) Cre-mediated site-specific recombination in zebrafish embryos. Dev Dyn 233(4):1366–1377PubMedCrossRefGoogle Scholar
  52. Thummel R, Burket CT, Hyde DR (2006b) Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration. TSW Develop Embryol 6:65–81Google Scholar
  53. Tomasiewicz HG, Flaherty DB, Soria JP, Wood JG (2002) Transgenic zebrafish model of neurodegeneration. J Neurosci Res 70(6):734–745PubMedCrossRefGoogle Scholar
  54. Traver D (2004) Cellular dissection of zebrafish hematopoiesis. Methods Cell Biol 76:127–149PubMedCrossRefGoogle Scholar
  55. Udvadia AJ, Linney E (2003) Windows into development: historic, current, and future perspectives on transgenic zebrafish. Dev Biol 256(1):1–17PubMedCrossRefGoogle Scholar
  56. Vihtelic TS, Hyde DR (2000) Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina. J Neurobiol 44(3):289–307PubMedCrossRefGoogle Scholar
  57. Vihtelic TS, Yamamoto Y, Springer SS, Jeffery WR, Hyde DR (2005) Lens opacity and photoreceptor degeneration in the zebrafish lens opaque mutant. Dev Dyn 233(1):52–65PubMedCrossRefGoogle Scholar
  58. Vihtelic TS, Yamamoto Y, Sweeney MT, Jeffery WR, Hyde DR (2001) Arrested differentiation and epithelial cell degeneration in zebrafish lens mutants. Dev Dyn 222(4):625–636PubMedCrossRefGoogle Scholar
  59. Westerfield M (1995) The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). University of Oregon Press, EugeneGoogle Scholar
  60. Whitehead GG, Makino S, Lien CL, Keating MT (2005) fgf20 is essential for initiating zebrafish fin regeneration. Science 310(5756):1957–1960PubMedCrossRefGoogle Scholar
  61. Yu CJ, Gao Y, Li P, Li L (2007) Synchronizing multiphasic circadian rhythms of rhodopsin promoter expression in rod photoreceptor cells. J Exp Biol 210:676–684PubMedCrossRefGoogle Scholar
  62. Zhang J, Bai S, Zhang X, Nagase H, Sarras MP Jr (2003) The expression of novel membrane-type matrix metalloproteinase isoforms is required for normal development of zebrafish embryos. Matrix Biol 22(3):279–293PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Christopher T. Burket
    • 1
  • Jacob E. Montgomery
    • 1
  • Ryan Thummel
    • 1
  • Sean C. Kassen
    • 1
  • Matthew C. LaFave
    • 1
  • David M. Langenau
    • 2
  • Leonard I. Zon
    • 2
    • 3
  • David R. Hyde
    • 1
    Email author
  1. 1.Department of Biological Sciences and the Center for Zebrafish ResearchUniversity of Notre DameNotre DameUSA
  2. 2.HHMI/Children’s Hospital of Boston, Harvard Medical SchoolBostonUSA
  3. 3.Stem Cell Program and Division Hematology/Oncology Children’s Hospital and Dana Farber Cancer Institute, Howard Hughes Medical InstituteHarvard Stem Cell Institute, Harvard Medical SchoolBostonUSA

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