Transgenic Research

, Volume 11, Issue 3, pp 279–289 | Cite as

An ES-like cell line from the marine fish Sparus aurata: Characterization and chimaera production

  • Julia Béjar
  • Yunhan Hong
  • M. Carmen AlvarezEmail author


Embryonic stem (ES) cells provide a unique tool for cell-mediated gene transfer and targeted gene mutations due to the possibility of in vitro selection of desired genotypes. When selected cells contribute to the germ line in chimaeric embryos, transgenic animals may be generated with modified genetic traits. Though the ES cell approach has up to now been limited to mice, there is an increasing interest to develop this technology in both model and commercial fish species, with so far promising results in the medaka and zebrafish. In this study, we present evidence regarding a long-term stable cell line (SaBE-1c), derived from embryonic cells of the aquaculture marine fish Sparus aurata which has been characterized for (i) cell proliferation, (ii) chromosome complement, (iii) molecular markers, and (iv) in vitro tests of pluripotency by alkaline phosphatase (AP) staining, telomerase activity, and induced cell differentiation. These cells have proved their pluripotent capacities by in vitro tests. Furthermore, we have demonstrated their ability to produce chimaeras and to contribute to the formation of tissues from all three embryonic germ layers. These features suggest that SaBE-1c cells have the potential for multiple applications for the ES technology in fish, with the added value of originating from an economically important species.

chimaeras ES – embryonic stem cells seabream Sparus aurata transgenic fish 


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  1. Alvarez MC, Otis J, Amores A and Guise K (1991) Short-term cell culture technique for obtaining chromosomes in marine and freshwater fish. J Fish Biol 39: 817–824.Google Scholar
  2. Armstrong L, Lako M, Lincoln J, Cairns PM and Hole N (2000) mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells. Mech Dev 97: 109–116.Google Scholar
  3. Batargias C, Dermitzakis E, Magoulas A and Zouros E (1999) Characterisation of six polymorphic microsatellite markers in gilt-head seabream, Sparus aurata (Linaeus 1758). Mol Ecol 8: 897–898.Google Scholar
  4. Béjar J, Hong Y and Alvarez MC (1999) Towards obtaining ES cells in the marine fish species Sparus aurata; multipassage maintenance, characterisation and transfection. Genet Anal 15: 125–129.Google Scholar
  5. Blackburn E (2000) Telomere states and cell fates. Nature 408: 53–56.Google Scholar
  6. Capecchi MR (2000) How close are we to implementing gene targeting in animals other than mouse? Proc Natl Acad Sci USA 97: 956–957.Google Scholar
  7. Collodi P, Kamei Y, Sharps A, Weber D and Barnes D (1992) Fish embryo cell cultures for derivation of stem cells and transgenic chimeras. Mol Mar Biol Biotechnol 1: 257–265.Google Scholar
  8. Evans MJ and Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156.Google Scholar
  9. Fléchon JE (1997) What are ES cells? In: Houdebine LM (ed.), Transgenic Animals Generation and Use. Part II, Section F, Harwood and Publ, Amsterdam.Google Scholar
  10. García-Pozo S, Béjar J, Shaw M and Alvarez MC (1998) Effect of exogenous DNA microinjection on early development response of the seabream (Sparus aurata) chimeras. Mol Mar Biol Biotechnol 7: 248–258.Google Scholar
  11. Hackett PB and Alvarez MC (2000) The molecular genetics of transgenic fish. In: Fingerman M and Nagabhushanam R (eds), Recent Advances in Marine Biotechnology. Vol 4, Aquaculture, Fishes (pp 77–145) Science Publisher, Enfield, USA.Google Scholar
  12. Hanazono Y, Yu JM, Dunbar CE and Emmons RV (1997) Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage. Hum Gene Ther 8: 11 1313–1319.Google Scholar
  13. Holt SE, Wright WE and Shay JW (1996) Regulation of telomerase activity in immortal cell lines. Mol Cell Biol 16: 2932–2939.Google Scholar
  14. Hong Y and Schartl M (1996) Establishment and growth responses of early medakafish (Oryzias latipes) embryonic cells in feeder layer-free cultures. Mol Mar Bio Biotechnol 5: 93–104.Google Scholar
  15. Hong Y, Winkler C and Schartl M (1996) Pluripotency and differentiation of embryonic stem cell lines from the medaka fish (Oryzias latipes). Mech Dev 60: 33–44.Google Scholar
  16. Hong Y, Winkler C and Schartl M(1998a) Production of medakafish chimaeras from a stable embryonic stem cell line. Proc Natl Acad Sci USA 95: 3679–3684.Google Scholar
  17. Hong Y, Winkler C and Schartl M (1998b) Efficiency of cell culture derivation from blastula embryos and chimaera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number. Dev Genes Evol 208: 595–602.Google Scholar
  18. Huang WY, Aramburu J, Douglas PS and Izumo S (2000) Transgenic expression of green fluorescent protein can cause dilated cardiomyopathy. Nat Med 6: 482–483.Google Scholar
  19. Longo L, Bygrave A, Grosveld FG and Pandolfi PP (1997) The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res 6: 321–328.Google Scholar
  20. Ma C, Fan L, Ganassin R, Bols N and Collodi P (2001) Production of zebrafish germ-line chimeras from embryo cell cultures. Proc Natl Acad Sci USA 98: 2461–2466.Google Scholar
  21. Mantell LL and Greider GW (1994) Telomerase activity in germ line and embryonic cells of Xenopus. EMBO J 13: 3211–3217.Google Scholar
  22. Nichols J, Evans EP and Smith AG (1990) Establishment of germ-line competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 110: 1341–1348.Google Scholar
  23. Pain B, Clark ME, Shen M, Nakazama H, Sakurai M, Samarut J et al. (1996) Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development 122: 2339–2348.Google Scholar
  24. Prelle K, Vassiliev IM, Vassilieva SG, Wolf E and Wobus AM (1999) Establishment of pluripotent cell lines from vertebrate species-present status and future prospects. Cells Tissues Organs 165: 220–236.Google Scholar
  25. Pyatiszek MA, Kim NW, Weinrich SL, Hiyama K, Hiyama E, Wright WE et al. (1995) Detection of telomerase activity in human cells and tumours by a telomeric repeat amplification protocol (TRAP). Meth Cell Sci 17: 1–15.Google Scholar
  26. Robertson EJ (1987) Embryo-derived stem cell lines. In: Robertson EJ (ed.), Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (pp: 71–112) IRL Press, Oxford.Google Scholar
  27. Shiels PG, Kind AJ, Campbell KH, Waddington D, Wilmut I, Colman A et al. (1999) Analysis of telomere lengths in cloned sheep. Nature 399: 316–317.Google Scholar
  28. Sola L and Cataudella S (1978) Prime observazioni sulla cariologia degli Sparidi Mediterranei. Boll Zool 45: 242.Google Scholar
  29. Sun L, Bradford CS, Ghosh C, Collodi P and Barnes DW (1995a) ES-like cell cultures derived from early zebrafish embryos. Mol Mar Biol Biotechnol 4: 193–199.Google Scholar
  30. Sun L, Bradford CS and Barnes DW (1995b) Feeder cell cultures for zebrafish embryonal cells in vitro. Mol Mar Biol Biotechnol 4: 43–50.Google Scholar
  31. Walsh S, Metzger D and Higuchi R (1991) Chelex-100 as a medium for simple extraction of DNA for PCR-based typing from forensic. Mater Bio Techniq 10(4): 506–513.Google Scholar
  32. Wakamatsu Y, Ozato K and Sasao T (1994) Establishment of a pluri-potent cell line derived from medaka (Oryzias latipes) blastula embryo. Mol Mar Biol Biotechnol 3: 185–191.Google Scholar
  33. Wobus AM, Holzhausen H, Jäkel P and Schöneich J (1984) Characterisation of a pluripotent stem cell line derived from a mouse embryo. Exp Cell Res 152: 212–219.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  1. 1.Departamento de Biología Celular y Genética, Facultad de CienciasUniversidad de MálagaMálagaSpain
  2. 2.Physiological Chemistry IBiocenter of the University of Würzburg, Am HublandWürzburgGermany

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