Abstract
Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an “all fish” gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is regulated by the 5′ promoter and 3′ termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1α) integrated as a single functional copy into a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1α integrant was organized as follows: base pairs 1580–2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete ocean pout antifreeze 3′ region, and the first 1678 bp of the ocean pout antifreeze 5′ region. Sequence analysis of the EO-1α integrant and genomic flanking regions in F2 and F4 generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct. These results indicate that the EO-1α transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Deitch EJ, Fletcher GL, Peterson LH, Costa LASF, Shears MA, Driedzic WR, Gamperl AK (2006) Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon (Salmo salar). J Exp Biol DOI: 10.1242/jeb.02105
Devlin RH, Yesaki TY, Biagi CA, Donaldson EM, Swansen P, Chan WK (1994) Extraordinary salmon growth. Nature 371:209–210
Devlin RH (1997) Transgenic Salmonids. In: Houdebine LM (ed) Transgenic animals, generation and use. Harwood Academic Publishers, pp105–117
Devlin RH, Biagi CA, Yesaki TY, Smailus DE, Byatt JC (2001) Growth of domesticated transgenic fish. Nature 409:781–782
Du SJ, Gong Z, Tan CH, Fletcher GL, Hew CL (1992a) Development of an all-fish gene cassette for gene transfer in aquaculture. Mol Mar Biol Biotech 1:290–300
Du SJ, Gong ZY, Fletcher GL, Shears MA, King MJ, Idler DR, Hew CL (1992b) Growth enhancement in transgenic Atlantic salmon by the use of an “all fish” chimeric growth hormone gene construct. Bio/Technol (NY) 10:176–181
Du SJ (1993) The isolation and characterization of chinook salmon GH genes and the creation of fast-growing Atlantic salmon by GH gene transfer. PhD Thesis, Department of Biochemistry, University of Toronto
Dunham RA, Chitmanat C, Nichols A, Argue B, Powers DA, Chen TT (1999) Predator avoidance of transgenic channel catfish containing salmonid growth hormone genes. Mar Biotechnol (NY) 1:545–551
Environment Canada (2005) Guidelines for the notification and testing of new substances: organisms. Section 4 technical information requirements. http://www.ec.gc.ca/substances/nsb/bioguide/eng/Bioguide_e.htm
FAO (1999) The state of world fisheries and aquaculture 1998. Food and Agriculture Organization, Rome
FAO (2003) FAO/WHO Expert consultation on the safety assessment of foods derived from genetically modified animals, including fish, Rome, 17–21 November, 2003
Fletcher GL, Davies PL (1991) Transgenic fish for aquaculture. In: Setlow JK (ed) Genetic engineering, principles and methods, vol. 13. Plenum Press, New York, pp331–370
Fletcher GL, SV Goddard, MA Shears, Sutterlin A, Hew CL (2001) Transgenic salmon: potential and hurdles. In: Toutant JP, Balazs, E (eds) Proceedings OECD symposium on “Molecular Farming,” pp 57–65
Fletcher GL, Shears MA, Yaskowiak E, King MJ, Goddard SV (2004) Gene transfer: potential to enhance the genome of Atlantic Salmon for aquaculture. Aust J Exp Ag 44:1095–1100
Gjedrem T (1997) Selective breeding to improve aquaculture production. World Aquacult 28:33–45
Gong Z, Vielkind JR, Hew CL (1991) Functional analysis and temporal expression of promoter regions from fish antifreeze protein genes in transgenic Japanese medaka embryos. Mol Mar Biol Biotech 1:64–72
Gong Z, Hew CL (1995) Transgenic fish in aquaculture and developmental biology. Curr Top Dev Biol 30:177–214
Hew CL, Wang NC, Joshi S, Fletcher GL, Scott GK, Hayes PH, Buettner B, Davies PL (1988) Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus. J Biol Chem 263:12049–12055
Hew CL, Trinh KY, Du SJ, Song S (1989) Molecular cloning and expression of salmon pituitary hormones. Fish Physiol Biochem 7:375–380
Hew CL, Fletcher GL (1996) Transgenic salmonid fish expressing exogenous salmonid growth hormone. US Patent No. 5,545,808
Hew CL, Du SJ, Gong Z, Shears MA, King MJ, Fletcher GL, Davies PL, Saunders RL (1998) Use of the fish antifreeze protein gene promoter in the production of growth hormone-transgenic salmon with enhanced growth performance. In: Altman A (ed) Biotechnology in Aquaculture. Marcel Dekker, Inc., pp 549–561
Hew CL, Fletcher GL (2001) Gene construct for production of transgenic fish. European Patent number 0,578,653, B1
Houdebine LM, Chourrout D (1991) Transgenesis in fish. Experentia 47:891–897
Joint Subcommittee on Aquaculture (1992) Aquaculture in the United States: status, opportunities, and recommendations. A report to the federal coordinating council on science, engineering and technology. Available from the U.S. Dept. of Agriculture, Office of Aquaculture, Washington, DC
Kochhar HS (2004) Notification guidelines for the environmental assessment of biotechnology—derived livestock animals. Canadian Food Inspection Agency, Animal Health and Production Devision, Veterinary Biologics Section. http://www.inspection.gc.ca/english/anima/vetbio/abu/biotech/guidedirecte.shtml
Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95(12):7203–7208
Liao J, Chan CH, Gong Z (1997) An alternative linker-mediated polymerase chain reaction method using a dideoxynucleotide to reduce amplification background. Anal Biochem 253:137–139
Martinez R, Estrada MP, Berlanga J, Guillen I, Hernandez O, Cabrera E, Pimentel R, Morales R, Herrera F, Morales A, Pina JC, Abad Z, Sanchez V, Melamed P, Lleonart R, de la Fuente J (1996) Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone. Mol Mar Biol Biotechnol 1:62–70
Nam Y, Noh J, Cho Y, Cho H, Cho K, Kim C, Kim D (2001) Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis. Transgenic Res 10:353–362
New MB (1997) Aquaculture and the capture fisheries—balancing the scales. World Aquacult 28:11–30
Pauly D, Christensen V, Dalsgaard J, Froese R, Torres F Jr (1998) Fishing down marine food webs. Science 279:860–863
Pitkanen TI, Krasnov A, Teerijoki H, Molsa H (1999) Transfer of growth hormone (GH) transgenes into Arctic charr. (Salvelinus alpinus L.) I. Growth response to various GH constructs. Genet Anal 15:91–98
Rahman MA, Ronyai A, Engidaw BZ, Jauncey K, Hwang G, Smith A, Roderick E, Pennman D, Varadi L, Maclean N (2001) Growth and nutrition trials of transgenic Nile tilapia containing an exogenous fish growth hormone gene. J Fish Biol 59:62–78
Rise ML, von Schalburg KR, Brown GD, Mawer MA, Devlin RH, Kuipers N, Busby M, Beetz-Sargent M, Alberto R, Gibbs AR, Hunt P, Shukin R, Zeznik JA, Nelson C, Jones SR, Smailus DE, Jones SJ, Schein JE, Marra MA, Butterfield YS, Stott JM, Ng SH, Davidson WS, Koop BF (2004) Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybridization characteristics. Genome Res 14:478–490
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, NY
U.S. FDA (1995) Points to consider in the manufacture and testing of therapeutic products for human use derived from transgenic animals. U.S. Food and Drug Administration, Center for Biologics Evaluation and Research. http://www.fda.gov/cber/gdlns/ptc_tga.txt
Watson R, Pauly D (2001) Systematic distortions in world fisheries catch trends. Nature 414:534–536
Wu B, Sun YH, Wang YP, Wang YW, Zhu ZY (2004) Sequences of transgene insertion sites in transgenic F4 common carp. Transgenic Res 13:95–96
Wu B, Sun YH, Wang YW, Wang YP, Zhu ZY (2005) Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.). Cell Res 15:447–454
Zhu Z (1992) Generation of fast growing transgenic fish, methods and mechanisms. In: Hew CL, Fletcher GL (eds), Transgenic fish, World Scientific Publishing, Singapore, pp 92–119
Zhu ZY, Sun YH (2000) Embryonic and genetic manipulation of fish. Cell Res 10:17–27
Zbikowska HM (2003) Fish can be first-advances to fish transgenesis for applications. Transgenic Res 12:379–389
Author information
Authors and Affiliations
Corresponding author
Additional information
An erratum to this article is available at http://dx.doi.org/10.1007/s11248-006-9059-6.
Rights and permissions
About this article
Cite this article
Yaskowiak, E.S., Shears, M.A., Agarwal-Mawal, A. et al. Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic Salmon. Transgenic Res 15, 465–480 (2006). https://doi.org/10.1007/s11248-006-0020-5
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11248-006-0020-5