Transgenic Research

, Volume 20, Issue 6, pp 1217–1226 | Cite as

Zebrafish HSC70 promoter to express carp muscle-specific creatine kinase for acclimation under cold condition

  • Chih-Lu Wu
  • Ta-Hui Lin
  • Tien-Lin Chang
  • Hsi-Wen Sun
  • Cho-Fat Hui
  • Jen-Leih WuEmail author
Original Paper


Zebrafish (Danio rerio) is used as a model system for in vivo studies. To expand the research scope of physical, biochemical and physiological studies, a cold-tolerant model of zebrafish was developed. The common carp (Cyprinus carpio) muscle form of creatine kinase (CK, EC can maintain enzymatic activity at a temperature of around 15°C. However, a cold-inducible promoter of zebrafish, hsc 70 (heat shock protein 70 cognate), is able to increase the expression of gene product by 9.8 fold at a temperature of 16°C. Therefore, the carp CK gene was promoted by hsc 70 and transfected into zebrafish embryos. Resulting transgenic zebrafish survived and could maintain its swimming behavior at 13°C, which was not possible with the wild-type zebrafish. The swimming distance of the transgenic fish was 42% greater than that of the wild type at 13°C. This new transgenic fish model is ideal for studies of ectothermal vertebrates in low-temperature environments.


Creatine kinase Heat shock cognate Hsc 70 Cold Inducible promoter Zebrafish 



Creatine kinase


Heat shock protein 70 cognate


Heat shock protein


Muscle form creatine kinase

M1- (M2-, M3-)CK

Carp muscle form creatine kinase subisoform 1 (2, 3)

CKM1 (2, 3)

Coding sequence of carp muscle form creatine kinase subisoform 1 (2, 3)


Reverse transcription polymerase chain reaction


Quantitative reverse transcription polymerase chain reaction


Elongation factor 1 alpha




Cold inducible promoter


Enhanced green fluorecence protein


Hour post-fertilization



This work was supported by grants from Academia Sinica and the Council of Agriculture, Taiwan (Project No. 9521011700060106F125).

Supplementary material

Supplementary material 1 (WMV 8568 kb)

Supplementary material 2 (WMV 3450 kb)

11248_2011_9488_MOESM3_ESM.doc (20 kb)
Supplementary material 3 (DOC 20 kb)


  1. Ali KS, Dorgai L, Abraham M, Hermesz E (2003) Tissue- and stressor-specific differential expression of two hsc70 genes in carp. Biochem Biophys Res Commun 307:503–509PubMedCrossRefGoogle Scholar
  2. Anderson JV, Li QB, Haskell DW, Guy CL (1994) Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation. Plant Physiol 104:1359–1370PubMedCrossRefGoogle Scholar
  3. Bernal D, Donley JM, Shadwick RE, Syme DA (2005) Mammal-like muscles power swimming in a cold-water shark. Nature 437:1349–1352PubMedCrossRefGoogle Scholar
  4. Boutilier RG, Donohoe PH, Tattersall GJ, West TG (1997) Hypometabolic homeostasis in overwintering aquatic amphibians. J Exp Biol 200:387–400PubMedGoogle Scholar
  5. Carmona MC, Valmaseda A, Brun S, Vinas O, Mampel T, Iglesias R, Giralt M, Villarroya F (1998) Differential regulation of uncoupling protein-2 and uncoupling protein-3 gene expression in brown adipose tissue during development and cold exposure. Biochem Biophys Res Commun 243:224–228PubMedCrossRefGoogle Scholar
  6. Chang BE, Lin CY, Kuo CM (1999) Molecular cloning of a cold-shock domain protein, zfY1, in zebrafish embryo. Biochim Biophys Acta 1433:343–349PubMedCrossRefGoogle Scholar
  7. Chou MY, Hsiao CD, Chen SC, Chen IW, Liu ST, Hwang PP (2008) Effects of hypothermia on gene expression in zebrafish gills: upregulation in differentiation and function of ionocytes as compensatory responses. J Exp Biol 211:3077–3084PubMedCrossRefGoogle Scholar
  8. Freeman BC, Morimoto RI (1996) The human cytosolic molecular chaperones hsp90, hsp70 (hsc70), and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J 15:2969–2979PubMedGoogle Scholar
  9. Gracey AY, Fraser EJ, Li W, Fang Y, Taylor RR, Rogers J, Brass A, Cossins AR (2004) Coping with cold, An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate. Proc Natl Acad Sci USA 101:16970–16975PubMedCrossRefGoogle Scholar
  10. Guderley H (2004) Metabolic responses to low temperature in fish muscle. Biol Rev Camb Philosoph Soc 79:409–427CrossRefGoogle Scholar
  11. Hazel JR (1984) Effects of temperature on the structure and metabolism of cell membranes in fish. Am J Physiol 246:R460–R470PubMedGoogle Scholar
  12. Hazel JR (1993) Thermal biology. In: Evans DH (ed) The physiology of fishes. CRC press Inc. Salem, MA, USA., pp 438–446Google Scholar
  13. Hohfeld J (1998) Regulation of the heat shock conjugate Hsc70 in the mammalian cell: the characterization of the anti-apoptotic protein BAG-1 provides novel insights. Biol Chem 379:269–274PubMedGoogle Scholar
  14. Hung JJ, Cheng TJ, Chang MD, Chen KD, Huang HL, Lai YK (1998) nvolvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells. J Cell Biochem 71:21–35PubMedCrossRefGoogle Scholar
  15. Imamura S, Ojima N, Yamashita M (2003) Cold-inducible expression of the cell division cycle gene CDC48 and its promotion of cell proliferation during cold acclimation in Zebrafish cells. FEBS Lett 549:14–20PubMedCrossRefGoogle Scholar
  16. Itoi S, Kinoshita S, Kikuchi K, Watabe S (2003) Changes of carp FoF1-ATPase in association with temperature acclimation. Am J Physiol Regul Integr Comp Physiol 284:R153–R163PubMedGoogle Scholar
  17. Johnston IA, Sidell BD, Driedzic WR (1985) Force—velocity characteristics and metabolism of carp muscle fibres following temperature acclimation. J Exp Biol 119:239–249PubMedGoogle Scholar
  18. Ju Z, Dunham RA, Liu Z (2002) Differential gene expression in the brain of channel catfish (Ictalurus punctatus) in response to cold acclimation. Mol Genet Genomics 268:87–95PubMedCrossRefGoogle Scholar
  19. Knight H, Zarka DG, Okamoto H, Thomashow MF, Knight MR (2004) Abscisic acid induces CBF gene transcription and subsequent induction of cold-regulated genes via the CRT promoter element. Plant Physiol 135:1710–1717PubMedCrossRefGoogle Scholar
  20. Kristiansen E, Zachariassen KE (2005) The mechanism by which fish antifreeze proteins cause thermal hysteresis. Cryobiology 51:262–280PubMedCrossRefGoogle Scholar
  21. Krumschnabel G, Biasi C, Schwarzbaum PJ, Wieser W (1997) Acute and chronic effects of temperature, and of nutritional state, on ion homeostasis and energy metabolism in teleost hepatocytes. J Comp Physiol B 167:280–286PubMedCrossRefGoogle Scholar
  22. Kvamme BO, Skern R, Frost P, Nilsen F (2004) Molecular characterisation of five trypsin-like peptidase transcripts from the salmon louse (Lepeophtheirus salmonis) intestine. Int J Parasitol 34:823–832PubMedCrossRefGoogle Scholar
  23. Leandro NS, Gonzales E, Ferro JA, Ferro MI, Givisiez PE, Macari M (2004) Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress. Mol Reprod Dev 67:172–177PubMedCrossRefGoogle Scholar
  24. Lim JW, Kim KH, Kim H (2008) NF-kappaB p65 regulates nuclear translocation of Ku70 via degradation of heat shock cognate protein 70 in pancreatic acinar AR42 J cells. Int J Biochem Cell Biol 40:2065–2077PubMedCrossRefGoogle Scholar
  25. Liu Y, Zhao J, Liu J, Zhang H, Liu M, Xiao X (2008) Upregulation of the constitutively expressed HSC70 by KLF4. Cell Stress Chaperones 13:337–345PubMedCrossRefGoogle Scholar
  26. Lopez-Olmeda JF, Madrid JA, Sanchez-Vazquez FJ (2006) Light and temperature cycles as zeitgebers of zebrafish (Danio rerio) circadian activity rhythms. Chronobiol Int 23:537–550PubMedCrossRefGoogle Scholar
  27. Murakami S (2006) Stress resistance in long-lived mouse models. Exp Gerontol 41:1014–1019PubMedCrossRefGoogle Scholar
  28. Narberhaus F (1999) Negative regulation of bacterial heat shock genes. Mol Microbiol 31:1–8PubMedCrossRefGoogle Scholar
  29. Neven LG, Haskell DW, Guy CL, Denslow N, Klein PA, Green LG, Sliverman A (1992) Association of 70-kilodalton heat- shock cognate proteins with acclimation to cold. Plant Physiol 99:1362–1369PubMedCrossRefGoogle Scholar
  30. Park YM, Mivechi NF, Auger EA, Hahn GM (1994) Altered regulation of heat shock gene expression in heat resistant mouse cells. Int J Radiat Oncol Biol Phys 28:179–187PubMedCrossRefGoogle Scholar
  31. Rensing L, Ruoff P (2002) Temperature effect on entrainment, phase shifting, and amplitude of circadian clocks and its molecular bases. Chronobiol Int 19:807–864PubMedCrossRefGoogle Scholar
  32. Rinehart JP, Yocum GD, Denlinger DL (2000) Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Ssarcophaga crassipalpis. Insect Biochem Mol Biol 30:515–521PubMedCrossRefGoogle Scholar
  33. Rinehart JP, Li A, Yocum GD, Robich RM, Hayward SA, Denlinger DL (2007) Up-regulation of heat shock proteins is essential for cold survival during insect diapause. Proc Natl Acad Sci USA 104:11130–11137PubMedCrossRefGoogle Scholar
  34. Roussel D, Rouanet JL, Duchamp C, Barre H (1998) Effects of cold acclimation and palmitate on energy coupling in duckling skeletal muscle mitochondria. FEBS Lett 439:258–262PubMedCrossRefGoogle Scholar
  35. Shim JK, Jung DO, Park JW, Kim DW, Ha DM, Lee KY (2006) Molecular cloning of the heat-shock cognate 70 (Hsc70) gene from the two-spotted spider mite, Tetranychus urticae, and its expression in response to heat shock and starvation. Comp Biochem Physiol B Biochem Mol Biol 145:288–295PubMedCrossRefGoogle Scholar
  36. Snodgrass JW (1991) Winter kills of Tilapia melanotheron in coastal Southeast Florida, 1989. Florida Science 54:85–86Google Scholar
  37. Sonoda S, Fukumoto K, Izumi Y, Yoshida H, Tsumuki H (2006) Cloning of heat shock protein genes (hsp90 and hsc70) and their expression during larval diapause and cold tolerance acquisition in the rice stem borer, Chilo suppressalis Walker. Arch Insect Biochem Physiol 63:36–47PubMedCrossRefGoogle Scholar
  38. Storey KB (1997) Metabolic regulation in mammalian hibernation: enzyme and protein adaptations. Comp Biochem Physiol A Physiol 118:1115–1124PubMedCrossRefGoogle Scholar
  39. Sun HW, Hui CF, Wu JL (1998) Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp (Cyprinus carpio) striated muscle. J Biol Chem 273:33774–33780PubMedCrossRefGoogle Scholar
  40. Sun HW, Liu CW, Hui CF, Wu JL (2002) The carp muscle-specific sub-isoenzymes of creatine kinase form distinct dimers at different temperatures. Biochem J 368:799–808PubMedCrossRefGoogle Scholar
  41. Swank DM, Rome LC (2001) The influence of thermal acclimation on power production during swimming. II. Mechanics of scup red muscle under in vivo conditions. J Exp Biol 204:419–430Google Scholar
  42. Takayama S, Krajewski S, Krajewska M, Kitada S, Zapata JM, Kochel K, Knee D, Scudiero D, Tudor G, Miller GJ, Miyashita T, Yamada M, Reed JC (1998) Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines. Cancer Res 58:3116–3131PubMedGoogle Scholar
  43. Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM (1992) Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the ‘phosphocreatine circuit’ for cellular energy homeostasis. Biochem J 281:21–40PubMedGoogle Scholar
  44. Westerfield M (1995) “The Zebrafish Book, A Guide for the Laboratory Use of Zebrafish (Danio rerio)”. University of Oregon Press, Eugene, USAGoogle Scholar
  45. Wu CL, Liu CW, Sun HW, Chang HC, Huang CJ, Hui CF, Wu JL (2008) The Carp M1 Muscle-Specific Creatine Kinase subisoform is Adaptive to the Synchronized Changes in Body Temperature and Intracellular pH that occur in the common carp. J Fish Biol 73:2513–2526CrossRefGoogle Scholar
  46. Yeh FL, Hsu T (2002) Differential regulation of spontaneous and heat-induced HSP 70 expression in developing zebrafish (Danio rerio). J Exp Zool 293:349–359PubMedCrossRefGoogle Scholar
  47. Yocum GD (2001) Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle. J Insect Physiol 47:1139–1145PubMedCrossRefGoogle Scholar
  48. Zhang C, Guy CL (2006) In vitro evidence of Hsc70 functioning as a molecular chaperone during cold stress. Plant Physiol Biochem 44:844–850PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Chih-Lu Wu
    • 1
    • 2
  • Ta-Hui Lin
    • 1
  • Tien-Lin Chang
    • 1
  • Hsi-Wen Sun
    • 3
  • Cho-Fat Hui
    • 1
  • Jen-Leih Wu
    • 1
    • 2
    Email author
  1. 1.Institute of Cellular and Organismic BiologyTaipeiTaiwan
  2. 2.Institute of Microbiology and Biochemistry, National Taiwan UniversityTaipeiTaiwan
  3. 3.Institute of Biotechnology, National Taipei University of TechnologyTaipeiTaiwan

Personalised recommendations