Abstract
We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (# 1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.
Similar content being viewed by others
References
Blin N and Stafford DW (1976) A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 3: 2303–2308.
Chakrabartty A, Ananthanarayanan VS and Hew CL (1989) Structure-function relationships in a winter flounder antifreeze polypeptide. J Biol Chem 264: 11313–11316.
Chan SL, Fletcher GL and Hew CL (1993) Control of antifreeze protein gene expression in winter flounder. In: Mochachka and Mommsen (eds) Biochemistry and Molecular Biology of Fishes (pp. 293–305) Elsevier Science Publishers.
Chan SL, Miao M, Fletcher GL and Hew CL (1997) The role of CCAAT/enhancer-binding protein α and a protein that binds to the activator-protein-1 site in the regulation of liver-specific expression of the winter flounder antifreeze protein gene. Eur J Biochem 247: 44–51.
Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159.
Davies PL and Hew CL (1990) Biochemistry of fish antifreeze proteins. FASEB J 4: 2460–2468.
Davies PL and Sykes BD (1997) Antifreeze proteins. Curr Opin Structural Biol 7: 828–834.
Deng G, Andrews DW and Laursen RA (1997) Amino acid sequence of a new type of antifreeze protein from the longhorn sculpin Myoxocephalus octodecimspinosis. FEBs Lett 402: 17–20.
DeVries AL (1984) Role of glycopeptides and peptides in inhibition of crystallization of water in polar fishes. Phil Trans R Soc Lond B304: 575–588.
Gong Z, Fletcher GL and Hew CL (1992) Tissue distribution of fish antifreeze protein mRNAs. Can J Zool 70: 810–814.
Gong Z, Ewart KV, Hu Z, Fletcher GL and Hew CL (1996) Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretory signal and prosequences. J Biol Chem 271: 4106–4112.
Hew CL, Scott GK and Davies PL (1986) Molecular biology of antifreeze. In: HC Heller, XY Musacchia and LCH Wang (eds) Living in the Cold: Physiological and Biochemical Adaptation (pp. 117–123) Elsevier Press, New York.
Hew CL, Fletcher GL and Davies PL (1995) Transgenic salmon: tailoring the genome for food production. J Fish Biol 47: 1–19.
Iyengar A, Müller F and Maclean N (1996) Regulation and expression of transgenes in fish – a review. Transgenic Res 5: 147–166.
Liao J, Chan CH and Gong Z (1997) An alternative linker mediated polymerase chain reaction method using a dideoxynucleotide to reduce amplification background. Anal Biochem 253: 137–139.
Rancourt DE, Walker VK and Davies PL (1987) Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster. Mol Biol 7: 2188–2195.
Scott GK, Hew CL and Davies PL (1985) Antifreeze protein genes are tandemly linked and clustered in the genome of the winter flounder. Proc Natl Acad Sci USA 82: 2613–2617.
ShearsMA, Fletcher GL, Hew CL, Gauthier S and Davies PL (1991) Transfer, expression and stable inheritance of antifreeze protein genes in Atlantic salmon (Salmo salar). Marine Mol Biol Biotech 1: 58–63.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hew, C., Poon, R., Xiong, F. et al. Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene. Transgenic Res 8, 405–414 (1999). https://doi.org/10.1023/A:1008900812864
Issue Date:
DOI: https://doi.org/10.1023/A:1008900812864