Transgenic Research

, Volume 20, Issue 6, pp 1333–1355 | Cite as

Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena

  • Young Sun Cho
  • Sang Yoon Lee
  • Youn Kyoung Kim
  • Dong Soo Kim
  • Yoon Kwon NamEmail author
Original Paper


Marine medaka Oryzias dancena, a candidate model organism, represents many attractive merits as a material for experimental transgenesis and/or heterologous expression assay particularly in the field of ecotoxicology and developmental biology. In this study, cytoskeletal β-actin gene was characterized from O. dancena and the functional capability of its promoter to drive constitutive expression of foreign reporter protein was evaluated. The O. dancena β-actin gene possessed a conserved genomic organization of vertebrate major cytoplasmic actin genes and the bioinformatic analysis of its 5′-upstream regulatory region predicted various transcription factor binding motifs. Heterologous expression assay using a red fluorescent protein (RFP) reporter construct driven by the O. dancena β-actin regulator resulted in stunningly bright expression of red fluorescence signals in not only microinjected embryos but also grown-up transgenic adults. Although founder transgenics exhibited mosaic patterns of RFP expression, transgenic offspring in subsequent generations displayed a vivid and uniform expression of RFP continually from embryos to adults. Based on the blot hybridization assays, two transgenic lines established in this study were proven to possess high copy numbers of transgene integrants (approximately 240 and 34 copies, respectively), and the transgenic genotype in both lines could successfully be passed stably up to three generations, although the rate of transgene transmission in one of the two transgenic lines was significantly lower than expected Mendelian ratio. Significant red fluorescence color could be ubiquitously observable in all the tissues or organs of the transgenics. Quantitative real-time RT-PCR represented that the expression pattern of transgene under the regulation of β-actin promoter would resemble, in overall, the regulation of endogenous β-actin gene in adult tissues, although putative mechanism for competitive or independent regulation between transgene and endogenous gene could also be found in several tissues. Results from this study undoubtedly indicate that the O. dancena β-actin promoter would be powerful enough to fluorescently visualize most cell types in vivo throughout its whole lifespan. This study could be a useful start point for a variety of transgenic experiments with this species concerning the constitutive expression of living fluorescent color reporters and other foreign proteins.


β-actin Gene and promoter structure RFP Transgenic expression Marine medaka Oryzias dancena 



This study was supported by a research fund from the Ministry of Land, Transport and Maritime Affairs, Korea (Project # 20088033-1).

Supplementary material

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Supplementary material 1 (PDF 20980 kb)


  1. Anderson KV (2000) Toll signaling pathways in the innate immune response. Curr Opin Immunol 12:13–19PubMedCrossRefGoogle Scholar
  2. Arukwe A (2006) Toxicological housekeeping genes: do they really keep the house? Environ Sci Technol 40:7944–7949PubMedCrossRefGoogle Scholar
  3. Ashe HL, Monks J, Wijgerde M, Fraser P, Proudfood NJ (1997) Intergenic transcription and transinduction of the human β-globin locus. Genes Dev 11:2494–2509PubMedCrossRefGoogle Scholar
  4. Baup D, Fraga L, Pernot E, Acker AV, Vanherck AS, Breckpot K, Thielemans K, Schurmans S, Moser M, Leo O (2010) Variegation and silencing in a lentiviral-based murine transgenic model. Transgenic Res 19:399–414PubMedCrossRefGoogle Scholar
  5. Brooks C, Hwang G, Maclean N (2007) Transgene activity following somatic transgenesis in Nile tilapia (Oreochromis niloticus). J Fish Biol 70:234–247CrossRefGoogle Scholar
  6. Burket CT, Montgomery JE, Thummel R, Kassen SC, FaFave MC, Langenau DM, Zon LI, Hyde DR (2008) Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters. Transgenic Res 17:265–279PubMedCrossRefGoogle Scholar
  7. Chen X, Li VWT, Yu RMK, Cheng SH (2008) Choriogenin mRNA as a sensitive molecular biomarker for estrogenic chemicals in developing brackish medaka (Oryzias melastigma). Ecotoxicol Environ Saf 71:200–208PubMedCrossRefGoogle Scholar
  8. Chen H, Hu J, Yang J, Wang Y, Xu H, Jiang Q, Gong Y, Gu Y, Song H (2010) Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens. Aquat Toxicol 96:53–61PubMedCrossRefGoogle Scholar
  9. Cho YS, Lee SY, Kim KH, Kim SK, Kim DS, Nam YK (2009a) Gene structure and differential modulation of multiple rockbream (Oplegnathus fasciatus) hepcidin isoforms resulting from different biological stimulations. Dev Comp Immunol 33:46–58PubMedCrossRefGoogle Scholar
  10. Cho YS, Lee SY, Bang IC, Kim DS, Nam YK (2009b) Genomic organization and mRNA expression of manganese superoxide dismutase (Mn-SOD) from Hemibarbus mylodon. Fish Shellfish Immunol 27:571–576PubMedCrossRefGoogle Scholar
  11. Cho YS, Lee SY, Kim DS, Nam YK (2010) Spawning performance, embryonic development and early viability under different salinity conditions in a euryhaline medaka Species, Oryzias dancena. Korean J Ichthyol 22:25–33Google Scholar
  12. Chou CY, Horng LS, Tsai HJ (2001) Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation. Transgenic Res 10:303–315PubMedCrossRefGoogle Scholar
  13. Davis BP, MacDonald RJ (1988) Limited transcription of rat elastase I transgene repeats in transgenic mice. Genes Dev 2:13–22PubMedCrossRefGoogle Scholar
  14. Devgan V, Rao MRS, Seshagiri PB (2004) Impact of embryonic expression of enhanced green fluorescent protein on early mouse development. Biochem Biophys Res Commun 313:1030–1036PubMedCrossRefGoogle Scholar
  15. Dorer DR, Henikoff S (1997) Transgene repeat arrays interact with distant heterochromatin and cause silencing in cis and trans. Genetics 147:1181–1190PubMedGoogle Scholar
  16. Er-meng Y, Xing Y, Hai-ying W, Jun-jie B, Shi-ling X, Hai-hua L, Qing J (2010) Isolation of Tanichthys albonubes β actin gene and production of transgenic Tanichthys albonubes. Fish Physiol Biochem 36:173–180CrossRefGoogle Scholar
  17. Fernandes JMO, Mommens M, Hagen O, Babiak I, Solberg C (2008) Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol B 150:23–32PubMedCrossRefGoogle Scholar
  18. Filby AL, Tyler CR (2007) Appropriate ‘housekeeping’ genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol Biol 8:10PubMedCrossRefGoogle Scholar
  19. Geurts AM, Collier LS, Geurts JL, Oseth LL, Bell ML, Mu D, Lucito R, Godbout SA, Green LE, Lowe SW, Hirsch BA, Leinwand LA, Largaespada DA (2006) Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers. PLoS Genet 2:e156PubMedCrossRefGoogle Scholar
  20. Gibbs PD, Schmale MC (2000) GFP as a genetic marker scorable throughout the life cycle of transgenic zebrafish. Mar Biotechnol 2:107–125PubMedGoogle Scholar
  21. Gong Z, Ju B, Wan H (2001) Green fluorescent protein (GFP) transgenic fish and their applications. Genetica 111:213–225PubMedCrossRefGoogle Scholar
  22. Hackett PB, Alvarez MC (2000) The molecular genetics of transgenic fish. In: Fingerman M, Nagabhushanam R (eds) Recent advances in marine biotechnology, vol 4. Science Publishers, Enfield, pp 77–145Google Scholar
  23. Hamada K, Tamaki K, Sasado T, Watai Y, Kani S, Wakamatsu Y, Ozato K, Kinoshita M, Kohno R, Takagi S, Kimura M (1998) Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes). Mol Mar Biol Biotechnol 7:173–180PubMedGoogle Scholar
  24. Hsiao C-D, Tsai HJ (2003) Transgenic zebrafish with fluorescent germ cell: a useful tool to visualize germ cell proliferation and juvenile hermaphroditism in vivo. Dev Biol 262:313–323PubMedCrossRefGoogle Scholar
  25. Huang W-T, Hsieh J-C, Chiou M-J, Chen J-Y, Wu J-L, Kuo C-M (2008) Application of RNAi technology to the inhibition of zebrafish GtHα, FSHβ, and LHβ expression and to functional analyses. Zool Sci 25:614–621PubMedCrossRefGoogle Scholar
  26. Hwang GL, Azizur RM, Abdul RS, Sohm F, Farahmand H, Smith A, Brooks C, Maclean N (2003) Isolation and characterization of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter. Biochim Biophys Acta - Gene Struct Exp 1625:11–18CrossRefGoogle Scholar
  27. Inoue K, Takei Y (2003) Asian medaka fishes offer new models for studying mechanisms of seawater adaptation. Comp Biochem Physiol B 136:635–645PubMedCrossRefGoogle Scholar
  28. James J, Osinska H, Hewett TE, Kimball T, Klevitsky R, Witt S, Hall DG, Gulick J, Robbins J (1999) Transgenic over-expression of a motor protein at high levels results in severe cardiac pathology. Transgenic Res 8:9–22PubMedCrossRefGoogle Scholar
  29. Kang CK, Tsai SC, Lee TH, Hwang PP (2008) Differential expression of branchial Na +/K + -ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater. Comp Biochem Physiol A 151:566–575CrossRefGoogle Scholar
  30. Kim DS, Kim BS, Lee SJ, Park IS, Nam YK (2004) Comparative analysis of inherited patterns of the transgene in transgenic mud loach Misgurnus mizolepis lines carrying the CAT reporter gene. Fish Sci 70:201–210CrossRefGoogle Scholar
  31. Kim KY, Lee SY, Cho YS, Bang IC, Kim DS, Nam YK (2008) Characterization and phylogeny of two beta-cytoskeletal actins from Hemibarbus mylodon (Cyprinidae, Cypriniformes), a threatened fish species in Korea. DNA Seq 19:87–97PubMedGoogle Scholar
  32. Kong RYC, Giesy JP, Wu RSS, Chen EXH, Ghiang MWL, Lim PL, Yuen BBH, Yip BWP, Mok HOL, Au DWT (2008) Development of a marine fish model for studying in vivo molecular responses in ecotoxicology. Aquat Toxicol 86:131–141PubMedCrossRefGoogle Scholar
  33. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K, Sindelka R, Sjöback R, Sjögreen B, Strömbom L, Ståhlberg A, Zoric N (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95–125PubMedCrossRefGoogle Scholar
  34. Kumar A, Crawford K, Flick R, Klevitsky R, Lorenz JN, Bove KE, Robbins J, Lessard JL (2004) Transgenic overexpression of cardiac actin in the mouse heart suggests coregulation of cardiac, skeletal and vascular actin expression. Transgenic Res 13:531–540PubMedCrossRefGoogle Scholar
  35. Lee SY, Kim KH, Nam YK (2009) Molecular characterization of rockbream (Oplegnathus fasciatus) cytoskeletal β-actin gene and its 5′-upstream regulatory region. Fish Aquat Sci 12:90–97CrossRefGoogle Scholar
  36. Lin C-Y, Yang P-H, Kao C-L, Huang H-I, Tsai H-J (2010) Transgenic zebrafish eggs containing bactericidal peptide is a novel food supplement enhancing resistance to pathogenic infection in fish. Fish Shellfish Immunol 28:419–427PubMedCrossRefGoogle Scholar
  37. Liu Z, Moav B, Faras AJ, Guise KS, Kapuscinski AR, Hackett PB (1990) Functional analysis of elements affecting expression of the beta-actin gene of carp. Mol Cell Biol 10:3432–3440PubMedGoogle Scholar
  38. Liu Z, Moav B, Faras AJ, Guise KS, Kapuscinski AR, Hackett PB (1991) Importance of the CArG box in regulation of β-actin-encoding genes. Gene 108:211–217PubMedCrossRefGoogle Scholar
  39. Matzke MA, Mette MF, Matzke AJM (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol 43:401–415PubMedCrossRefGoogle Scholar
  40. McDermott BM, Asai Y, Baucom JM, Jani SD, Castellanos Y, Bomez G, McClintock JM, Starr CJ, Hudspeth AJ (2010) Transgenic labeling of hair cells in the zebrafish acousticolateralis system. Gene Exp Patterns 10:113–118CrossRefGoogle Scholar
  41. Mitter K, Kotoulas G, Magoulas A, Mulero V, Sepulcre P, Figueras A, Novoa B, Sarropoulou E (2009) Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax). Comp Biochem Physiol B 153:340–347PubMedCrossRefGoogle Scholar
  42. Miwa T, Manabe Y, Kurokawa K, Kamada S, Kanda N, Bruns G, Ueyama H, Kakunaga T (1991) Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes. Mol Cell Biol 11:3296–3306PubMedGoogle Scholar
  43. Nam YK, Noh CH, Kim DS (1999) Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach (Misgurnus mizolepis). Aquaculture 172:229–245CrossRefGoogle Scholar
  44. Nam YK, Noh JK, Cho YS, Cho HJ, Cho KN, Kim CG, Kim DS (2001) Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis. Transgenic Res 10:353–362PubMedCrossRefGoogle Scholar
  45. Nam YK, Park JE, Kim KK, Kim DS (2003) A rapid and simple PCR-based method for analysis of transgenic fish using a restricted amount of fin tissue. Transgenic Res 12:523–525PubMedCrossRefGoogle Scholar
  46. Nam YK, Maclean N, Fu C, Pandian TJ, Eguia MRR (2007) Development of transgenic fish: scientific background. In: Kapuscinski AR, Hayes KR, Li S, Dana G (eds) Environmental risk assessment for genetically modified organisms, vol 3. CABI Press, Cambridge, pp 61–94Google Scholar
  47. Nam YK, Maclean N, Hwang G, Kim DS (2008) Autotransgenic and allotransgenic manipulation of growth traits in fish for aquaculture: a review. J Fish Biol 72:1–26CrossRefGoogle Scholar
  48. Noh JK, Cho KN, Han EH, Kim A, Lee JS, Kim DS, Kim CG (2003) Genomic cloning of mud loach, Misgurnus mizolepis (Cypriniformes, Cobitidae) beta-actin gene and usefulness of its promoter region for fish transgenesis. Mar Biotechnol 5:244–252PubMedCrossRefGoogle Scholar
  49. Pan X, Zhan H, Gong Z (2008) Ornamental expression of red fluorescent protein in transgenic founders of white skirt tetra (Gymnocorymbus ternetzi). Mar Biotechnol 10:497–501PubMedCrossRefGoogle Scholar
  50. Patil JG, Wong V, Khoo HW (1994) Assessment of pMTL construct for detection in vivo of luciferase expression and fate of the transgene in the zebrafish, Brachydanio rerio. Zool Sci 11:63–68PubMedGoogle Scholar
  51. Rahman MA, Hwang GL, Razak SA, Shom F, Maclean N (2000) Copy number related transgene expression and mosaic somatic expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus). Transgenic Res 9:417–427PubMedCrossRefGoogle Scholar
  52. Reece KS, McElroy D, Wu R (1992) Function and evolution of actins. Evol Biol 26:1–34CrossRefGoogle Scholar
  53. Ruiz S, Tafalla C, Cuesta A, Estepa A, Coll JM (2008) In vitro search for alternative promoters to the human immediate early cytomegalovirus (IE-CMV) to express the G gene of viral haemorrhagic septicemia virus (VHSV) in fish epithelial cells. Vaccine 26:6620–6629PubMedCrossRefGoogle Scholar
  54. Scholer H, Haslinger A, Heguy A, Holtgreve H, Karin M (1986) In vivo competition between a metallothionein regulatory element and the SV40 enhancer. Science 232:76–80PubMedCrossRefGoogle Scholar
  55. Small BC, Murdock CA, Bilodeau-Bourgeois AL, Peterson BC, Waldbieser GC (2008) Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions. Comp Biochem Physiol B 151:296–304PubMedCrossRefGoogle Scholar
  56. Song HY, Nam YK, Bang IC, Kim DS (2009) Embryogenesis and early ontogenesis of a marine medaka, Oryzias dancena. Korean J Ichthyol 21:227–238Google Scholar
  57. Tafalla C, Estepa A, Coll JM (2006) Fish transposons and their potential use in aquaculture. J Biotechnol 123:397–412PubMedCrossRefGoogle Scholar
  58. Takagi S, Sasado T, Tamiya G, Ozato K, Wakamatsu Y, Takeshita A, Kimura M (1994) An efficient expression vector for transgenic medaka construction. Mol Mar Biol Biotechnol 3:192–199PubMedGoogle Scholar
  59. Thermes V, Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly JS (2002) I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech Dev 118:91–98PubMedCrossRefGoogle Scholar
  60. Truksa J, Lee P, Beutler E (2007) The role of STAT, AP-1, E-box and TIEG motifs in the regulation of hepcidin by IL-6 and BMP-9: lessons from human HAMP and murine Hamp1 and Hamp2 gene promoters. Blood Cells Mol Dis 39:255–262PubMedCrossRefGoogle Scholar
  61. Tsai HJ, Wang SH, Inoue K, Takagi S, Kimura M, Wakamatsu Y, Ozato K (1995) Initiation of the transgenic lacZ gene expression in medaka (Oryzias latipes) embryos. Mol Mar Biol Biotechnol 4:1–9PubMedGoogle Scholar
  62. Uh M, Khattra J, Devlin RH (2006) Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA. Transgenic Res 15:711–727PubMedCrossRefGoogle Scholar
  63. Uzbekova S, Amoros C, Cauty C, Mambrini M, Perrot E, Hew CL, Chourrout D, Prunet P (2003) Analysis of cell-specificity and variegation of transgene expression driven by salmon prolactin promoter in stable lines of transgenic rainbow trout. Transgenic Res 12:213–227PubMedCrossRefGoogle Scholar
  64. Venkatesh B, Tay BH, Elgar G, Brenner S (1996) Isolation, characterization and evolution of nine pufferfish (Fugu rubripes) actin genes. J Mol Biol 259:655–665PubMedCrossRefGoogle Scholar
  65. Venugopal T, Anathy V, Kirankumar S, Pandian TJ (2004) Growth enhancement and food conversion efficiency of transgenic fish Labeo rohita. J Exp Zool 301:477–490CrossRefGoogle Scholar
  66. Williams DW, Müller F, Lavender FL, Orban L, Maclean N (1996) High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish (Danio rerio) embryos. Transgenic Res 5:433–442PubMedCrossRefGoogle Scholar
  67. Xie J, Lü L, Deng M, Weng S, Zhu J, Wu Y, Gan L, Chan SM, He J (2005) Inhibition of reporter gene and Iridovirus-tiger frog virus in fish cell by RNA interference. Virology 338:43–52PubMedCrossRefGoogle Scholar
  68. Zeng Z, Shan T, Tong Y, Lam SH, Gong Z (2005) Development of estrogen-responsive transgenic medaka for environmental monitoring of endocrine disrupters. Environ Sci Technol 39:9001–9008PubMedCrossRefGoogle Scholar
  69. Zenke K, Nam YK, Kim KH (2010) Development of siRNA expression vector utilizing rock bream β-actin promoter: a potential therapeutic tool against viral infection in fish. Appl Microbiol Biotechnol 85:679–690PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Young Sun Cho
    • 1
  • Sang Yoon Lee
    • 2
  • Youn Kyoung Kim
    • 1
  • Dong Soo Kim
    • 3
  • Yoon Kwon Nam
    • 3
    Email author
  1. 1.Institute of Marine Living Modified OrganismsPukyong National UniversityBusanKorea
  2. 2.Department of Fisheries BiologyPukyong National UniversityBusanKorea
  3. 3.Department of Marine Bio-Materials and AquaculturePukyong National UniversityBusanKorea

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