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Towards obtaining ES cells in the marine fish species Sparus aurata; multipassage maintenance, characterization and transfection
Genetic Analysis: Biomolecular Engineering 15 (1999) 125 – 129 www.elsevier.com/locate/gat
Towards obtaining ES cells in the marine fish species Sparus aurata; multipassage maintenance, characterization and transfection J. Be´jar a, Y. Hong b, M.C. Alvarez a,* b
a Departamento de Gene´tica, Facultad de Ciencias, Uni6ersidad de Ma´laga, 29071 Ma´laga, Spain Physiological Chemistry I, Biocenter of the Uni6ersity of Wu¨rzburg, Am Hubland 97074 Wu¨rzburg, Germany
Abstract Animal Embryonic-Stem (ES) cells represents a unique tool in animal genetic manipulation. Though putative ES cells from several species have been reported, only those from mice proved successful. In this work, a long-term embryonic cell culture, derived from the commercial fish (Sparus aurata), is reported. These cells have been in vitro characterized for totipotency and transfected with a GFP plasmid. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Sparus aurata; Seabream; Embryonic stem cells; Transgenic fish.
Embryonic stem (ES) cells derive from the culture of animal embryonic cells and remain totipotent under appropriate conditions [1,2]. When transplanted into recipient embryos, they produce chimeras and can contribute to any tissue including the germline. Fertilization of ES cell- gametes will result in whole individuals with traits of the ES cells. A combination of gene transfer into ES cells and production of chimeras can generate transgenic animals. Currently, DNA transfer in animals occurs at low efficiency. In contrast, making transgenics using ES cells allows checking for desired integration and expression of transgenes, (in vitro gene targeting). The phenotypic effects are detected in vivo after transferring cells to the recipient embryo. ES cells can also be used as in vitro model of differentiation and as source of totipotent nuclei for nuclear transfer. ES cell lines able to colonize the germ line, have so far been limited to mice [1 – 3]. Putative ES cell cultures have been reported in both mammalian [4] and nonmammalian species [5], although reproducible germline chimeras have not been described. * Corresponding author. Tel.: +34-5-2131967; fax: 2132000. E-mail address: [email protected] (M.C. Alvarez)
+34-5-
Initial attempts to obtain ES cells in zebrafish [6,7] were only partially successful. In medakafish however, a stable cell line showing in vitro totipotency traits [8] and high efficiency in forming chimeras [9] has been established. The stage is set for application of ES cell technology to commercial fish species to improve productivity by transgenesis. The marine fish Sparus aurata is very important in the Mediterranean aquaculture. The information generated around its biology, makes it as ‘model’ for domestication of related species, thus a project to establish ES cells from S. aurata has been initiated. Preliminary results on short-term embryonic cell cultures, were reported [10]. In this work we present the multipassage maintenance of an ES-like cell culture, exhibiting in vitro totipotency traits required for successful chimeras, and its ability to be transfected. To establish a seabream ES-like cell culture, we started from mid-blastula embryos of around 1000 cells (Fig. 1(a)) which were dechorionized with forceps. Cells in this stage proved successful in contributing to the germ line in both zebrafish [11] and medaka [12] chimeras. Once cultures were initiated (Fig. 1(b)), the main challenge was setting up conditions to allow longterm cultivation and preventing from spontaneous differentiation.
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J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129
The protocols applied to fish have been based on those set up for mouse cells, including a feeder layer of fibroblastic cells [1] or an inhibitory source of differentiation. However in our case medium composition and feeder free conditions established for medakafish [13], were generally adopted. All components were tested in a dose-dependent way for their efficiency in growth promotion and inhibition of cell differentiation [10] and results not presented). The optimal conditions for the seabream widely agreed with those of medaka, except for the incubation temperature (26°C), embryo extract and serum components, which supports the species-specific properties of cell regulating factors. Because the same feeder free conditions work for seabream as for medakafish, that are phylogenetically quite separated (in different orders), they should also be applicable to other fish species of commercial importance. Our study additionally strengthen the specific performance of fish ES cells when compared with other animal species, such as mammals [14] or chicken [5], in terms of not being dependent upon feeder layer cells or other inhibitory differentiation factors, which obviously represent disadvantages in the manipulation of ES cell cultures. Twenty-five primary cultures were initiated, about 50% started differentiation during the 10 first days (second-third passage) and eventually died. In this period a large variation in the success of different batches, was observed. The remainder cultures showed stable growth and typical morphology of undifferentiated cells (small size, round or polygonal shape, large nuclei). From these, most of them underwent differentiation and senescence changes around passage 9 – 10, thus indicating to be a critical stage in the culture progress. The differentiation tendency was strongly dependent upon culture conditions especially cell density; lower densities favoured cell differentiation. The cell types identified by morphological criteria were mostly fibroblastic-like (Fig. 1(e)) and less frequently neuron-like, muscle and pigmented cells (results not presented). From this set of cultures only one survived (SaBE1 after S. aurata blastula embryos), which has been cultured for about 9 months (45 passages), without apparent signs of instability or differentiation. It has been frozen and recultured several times without changes in morphology and growth. Colonies with a compact and round morphology have been formed in different passages (Fig. 1(c)), after plating at low densities (10–50 cells/cm2).
The endogenous alkaline phosphatase (AP) activity proved a valuable marker in putative ES cells of other animal species including fish [13,15] and was assayed at blastomeric cells and at different passages. In cultures with homogeneous cell population (typical ES-like morphology), all cells showed intensive staining (Fig. 1(d)). However in heterogeneous cultures (Fig. 1(e)), only cells with ES-like morphology were positive, while cells with differentiated phenotype were AP-negative. All cell colonies (Fig. 1(c)) showed a uniform and strong staining pattern (results not presented), thus indicating its undifferentiated state. For chromosome analysis, exponentially growing cultures from different passages were treated according to [16]. Chromosome euploidy is a requisite for ES cells to contribute to germ line in host chimera. The chromosome complement was assessed at passages 2, 18, 30 and 40. The respective counts are shown in Fig. 2. The modal group of metaphases with 2n= 48, showed the standard chromosome morphology of this species [17,18]. Chromosome values below and above the diploid number, can be artifacts inherent to the technique. In the four passages screened, the proportion of euploid metaphases (Fig. 2) is significant when compared with other seabream blastomere cultures with differentiated morphology, in which the distributions of the chromosome number presented an overhelming proportion of aneuploid metaphases mostly of poliploid origin (results not presented). Chromosome stability is a main property to be preserved in the ES cells. It is known in mice that the number of culture passages correlates negatively with their ability to contribute to the chimeras and that aneuploidy increases with longterm ES cell cultures. There are some indications [19] supporting the idea that chromosome changes and not the loss of totipotency, is the major reason for not colonizing all tissues including the germ line in the chimeras. It is well known that ES cell lines, like any long-term cell culture, are karyologically unstable and can drift towards an aneuploid modal distribution of chromosomes [20]. In mouse ES cells this is apparently favoured in feeder free systems [21]. We have however shown that in our SaBE1 cell culture, as well as in the medaka MES cell lines [8] the cells retain an apparently normal karyotype in absence of feeder cells. The in vitro characterization performed in the SaBE1 cell culture, reflects so far ES-like properties, thus looking very promising. This situation prompts us to ac-
Fig. 1. Cell culture from embryonic cells of S aurata. (a) Mid-blastula embryos at 6 h post-fertilization. A large lipidic drop and the blastomere mass are arrowheaded. Bar, 500 mm. (b) Single blastomeres in the culture dish soon after seeding. One blastomere is undergoing division (arrowhead). Bar, 50-mm. (c) Cell colony formed from passage 23 of the SaBE 1 culture, with round shape and smooth aspect. Bar, 50mm. (d) Homogeneous SaBE 1 cell culture from passage 20, showing typical morphology of ES cells and strong AP activity. Bar, 50mm. (e) Blastomeric cell culture from seabream at passage 6, showing smaller cells with ES-like morphology and intense AP staining, as well as larger cells apparently differentiated into fibroblastic-like type and without AP activity (arrowheads). Bar, 50mm. (e) Cells from the SaBE 1 culture at passage 12 showing a strong expression of the GFP gene marker, after being selected with the G418 drug. Bar, 50mm.
J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129
Fig. 1.
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J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129
Fig. 2. Chromosome number distribution of cells from SaBE 1 culture at passages 2, 8, 20 and 40. The n values (number of metaphases counted) corresponds to 21, 24, 26 and 30, respectively.
complish further tests of totipotency such as histochemical markers or telomerase activity, which proved very efficient in mouse [22] and chicken [5]. Additionally the in vitro differentiation into different cell types derived from all germ layers, will be induced. The final recognition as true ES cells would need however the in vivo proof of totipotency by injecting the cells into recipient embryos. The ability of these cells to be transfected is very important, either to introduce a desired mutation to be transmitted in a mendelian way, or as an in vivo gene marker, to check the contribution of ES cells to the chimeras. Cells were transfected with the pEGFP-N1 plasmid (Clontech Laboratories GmbH, Heidelberg, Germany), using a modification of the CaPO4 coprecipitation method [23]. The highest transfection efficiency was 1/4000 cells. The cells expressing GFP successfully responded to the positive selection by the drug G-418, after being kept for about 50 days under the selection conditions (Fig. 1(f)). Both the successful transformation of the SaBE1 cells with the pEGFP-N1 plasmid and its apparently efficient expression, represent important steps in the development process of ES-cells in S. aurata. They open the possibility for the homologous recombination process by using a suitable construction on one side and to check the fate of the ES-cells in the receptor embryo by means of the vital GFP marker, on the other. Actually genetic labeling with a GFP construct proved the most powerful approach for the in vivo screening chimeras of medaka [9].
Acknowledgements The authors acknowledge the Co. Cupimar, S.A. (San
Fernando, Ca´diz), for providing the eggs and the facilities. This work was supported by Grant BIO2-CT930430 from EU. References [1] Evans MJ, Kaufman MH. Nature 1981;292:154 – 6. [2] Martin GR. Proc Natl Acad Sci USA 1981;78:7634 – 8. [3] Bradley A, Evans M, Kaufman MH. Nature 1984;309:255– 6. [4] Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ. J Reprod Fertil 1991;43:255 – 60. [5] Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut J, Etches RJ. Development 1996;122:2339 – 48. [6] Collodi P, Kamei Y, Sharps A, Weber D, Barnes D. Mol Mar Biol Biotechnol 1992;1:257 – 65. [7] Sun L, Bradford CS, Ghosh C, Collodi P, Barnes DW. Mol Mar Biol Biotechnol 1995;4:193 – 9. [8] Hong Y, Winkler C, Schartl M. Mech Dev 1996;60:33–44. [9] Hong Y, Winkler C, Schartl M. Proc Natl Acad Sci USA 1998;95:3679 – 84. [10] Be´jar J, Hong Y, Alvarez MC. CIHEAM J Cah Options Mediterr 1998;34:131 – 9. [11] Lin S, Long W, Chen J, Hopkins N. Proc Natl Acad Sci USA 1992;89:4519 – 23. [12] Wakamatsu Y, Ozato K, Hashimoto H, Kinoshita M, Sakaguchi M, Iwamatsu T, Hyodo-Taguchi Y, Tomita H. Mol Mar Biol Biotechnol 1993;2:325 – 32. [13] Hong Y, Schartl M. Mol Mar Biol Biotechnol 1996;5:93– 104. [14] Flechon JE. In: Houbine LM, editor. Transgenic animals: Generation and use. Part II. Amsterdam: Harwood and Poubl, 1997:157 – 65 section F. [15] Wakamatsu Y, Ozato K, Sasado T. Mol Mar Biol Biotechnol 1994;3:185 – 91. [16] Alvarez MC, Otis J, Amores A, Guise K. J Fish Biol 1991;39:817 – 24. [17] Be´jar J, Borrego JJ, Alvarez MC. Aquaculture 1997;150:143– 53. [18] Sola L, Cataudella S. Boll Zool 1978;45:242.
J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129 [19] Longo L, Bygrave A, Grosveld FG, Pandolfi PP. Transgenic Res 1997;6:321 – 8. [20] Robertson EJ. In: Robertson EJ, editor. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: UK:IRL. Press, 1987:71 – 112.
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[21] Nichols J, Evans EP, Smith AG. Development 1990;110:1341–8. [22] Solter D, Knowles BB. Proc Natl Acad Sci USA 1978;75:5565– 9. [23] Friedenreich H, Schartl M. Nucl Acids Res 1990;18:3299– 305.
Towards obtaining ES cells in the marine fish species Sparus aurata; multipassage maintenance, characterization and transfection J. Be´jar a, Y. Hong b, M.C. Alvarez a,* b
a Departamento de Gene´tica, Facultad de Ciencias, Uni6ersidad de Ma´laga, 29071 Ma´laga, Spain Physiological Chemistry I, Biocenter of the Uni6ersity of Wu¨rzburg, Am Hubland 97074 Wu¨rzburg, Germany
Abstract Animal Embryonic-Stem (ES) cells represents a unique tool in animal genetic manipulation. Though putative ES cells from several species have been reported, only those from mice proved successful. In this work, a long-term embryonic cell culture, derived from the commercial fish (Sparus aurata), is reported. These cells have been in vitro characterized for totipotency and transfected with a GFP plasmid. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Sparus aurata; Seabream; Embryonic stem cells; Transgenic fish.
Embryonic stem (ES) cells derive from the culture of animal embryonic cells and remain totipotent under appropriate conditions [1,2]. When transplanted into recipient embryos, they produce chimeras and can contribute to any tissue including the germline. Fertilization of ES cell- gametes will result in whole individuals with traits of the ES cells. A combination of gene transfer into ES cells and production of chimeras can generate transgenic animals. Currently, DNA transfer in animals occurs at low efficiency. In contrast, making transgenics using ES cells allows checking for desired integration and expression of transgenes, (in vitro gene targeting). The phenotypic effects are detected in vivo after transferring cells to the recipient embryo. ES cells can also be used as in vitro model of differentiation and as source of totipotent nuclei for nuclear transfer. ES cell lines able to colonize the germ line, have so far been limited to mice [1 – 3]. Putative ES cell cultures have been reported in both mammalian [4] and nonmammalian species [5], although reproducible germline chimeras have not been described. * Corresponding author. Tel.: +34-5-2131967; fax: 2132000. E-mail address: [email protected] (M.C. Alvarez)
+34-5-
Initial attempts to obtain ES cells in zebrafish [6,7] were only partially successful. In medakafish however, a stable cell line showing in vitro totipotency traits [8] and high efficiency in forming chimeras [9] has been established. The stage is set for application of ES cell technology to commercial fish species to improve productivity by transgenesis. The marine fish Sparus aurata is very important in the Mediterranean aquaculture. The information generated around its biology, makes it as ‘model’ for domestication of related species, thus a project to establish ES cells from S. aurata has been initiated. Preliminary results on short-term embryonic cell cultures, were reported [10]. In this work we present the multipassage maintenance of an ES-like cell culture, exhibiting in vitro totipotency traits required for successful chimeras, and its ability to be transfected. To establish a seabream ES-like cell culture, we started from mid-blastula embryos of around 1000 cells (Fig. 1(a)) which were dechorionized with forceps. Cells in this stage proved successful in contributing to the germ line in both zebrafish [11] and medaka [12] chimeras. Once cultures were initiated (Fig. 1(b)), the main challenge was setting up conditions to allow longterm cultivation and preventing from spontaneous differentiation.
1050-3862/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 1 0 5 0 - 3 8 6 2 ( 9 9 ) 0 0 0 1 5 - 7
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J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129
The protocols applied to fish have been based on those set up for mouse cells, including a feeder layer of fibroblastic cells [1] or an inhibitory source of differentiation. However in our case medium composition and feeder free conditions established for medakafish [13], were generally adopted. All components were tested in a dose-dependent way for their efficiency in growth promotion and inhibition of cell differentiation [10] and results not presented). The optimal conditions for the seabream widely agreed with those of medaka, except for the incubation temperature (26°C), embryo extract and serum components, which supports the species-specific properties of cell regulating factors. Because the same feeder free conditions work for seabream as for medakafish, that are phylogenetically quite separated (in different orders), they should also be applicable to other fish species of commercial importance. Our study additionally strengthen the specific performance of fish ES cells when compared with other animal species, such as mammals [14] or chicken [5], in terms of not being dependent upon feeder layer cells or other inhibitory differentiation factors, which obviously represent disadvantages in the manipulation of ES cell cultures. Twenty-five primary cultures were initiated, about 50% started differentiation during the 10 first days (second-third passage) and eventually died. In this period a large variation in the success of different batches, was observed. The remainder cultures showed stable growth and typical morphology of undifferentiated cells (small size, round or polygonal shape, large nuclei). From these, most of them underwent differentiation and senescence changes around passage 9 – 10, thus indicating to be a critical stage in the culture progress. The differentiation tendency was strongly dependent upon culture conditions especially cell density; lower densities favoured cell differentiation. The cell types identified by morphological criteria were mostly fibroblastic-like (Fig. 1(e)) and less frequently neuron-like, muscle and pigmented cells (results not presented). From this set of cultures only one survived (SaBE1 after S. aurata blastula embryos), which has been cultured for about 9 months (45 passages), without apparent signs of instability or differentiation. It has been frozen and recultured several times without changes in morphology and growth. Colonies with a compact and round morphology have been formed in different passages (Fig. 1(c)), after plating at low densities (10–50 cells/cm2).
The endogenous alkaline phosphatase (AP) activity proved a valuable marker in putative ES cells of other animal species including fish [13,15] and was assayed at blastomeric cells and at different passages. In cultures with homogeneous cell population (typical ES-like morphology), all cells showed intensive staining (Fig. 1(d)). However in heterogeneous cultures (Fig. 1(e)), only cells with ES-like morphology were positive, while cells with differentiated phenotype were AP-negative. All cell colonies (Fig. 1(c)) showed a uniform and strong staining pattern (results not presented), thus indicating its undifferentiated state. For chromosome analysis, exponentially growing cultures from different passages were treated according to [16]. Chromosome euploidy is a requisite for ES cells to contribute to germ line in host chimera. The chromosome complement was assessed at passages 2, 18, 30 and 40. The respective counts are shown in Fig. 2. The modal group of metaphases with 2n= 48, showed the standard chromosome morphology of this species [17,18]. Chromosome values below and above the diploid number, can be artifacts inherent to the technique. In the four passages screened, the proportion of euploid metaphases (Fig. 2) is significant when compared with other seabream blastomere cultures with differentiated morphology, in which the distributions of the chromosome number presented an overhelming proportion of aneuploid metaphases mostly of poliploid origin (results not presented). Chromosome stability is a main property to be preserved in the ES cells. It is known in mice that the number of culture passages correlates negatively with their ability to contribute to the chimeras and that aneuploidy increases with longterm ES cell cultures. There are some indications [19] supporting the idea that chromosome changes and not the loss of totipotency, is the major reason for not colonizing all tissues including the germ line in the chimeras. It is well known that ES cell lines, like any long-term cell culture, are karyologically unstable and can drift towards an aneuploid modal distribution of chromosomes [20]. In mouse ES cells this is apparently favoured in feeder free systems [21]. We have however shown that in our SaBE1 cell culture, as well as in the medaka MES cell lines [8] the cells retain an apparently normal karyotype in absence of feeder cells. The in vitro characterization performed in the SaBE1 cell culture, reflects so far ES-like properties, thus looking very promising. This situation prompts us to ac-
Fig. 1. Cell culture from embryonic cells of S aurata. (a) Mid-blastula embryos at 6 h post-fertilization. A large lipidic drop and the blastomere mass are arrowheaded. Bar, 500 mm. (b) Single blastomeres in the culture dish soon after seeding. One blastomere is undergoing division (arrowhead). Bar, 50-mm. (c) Cell colony formed from passage 23 of the SaBE 1 culture, with round shape and smooth aspect. Bar, 50mm. (d) Homogeneous SaBE 1 cell culture from passage 20, showing typical morphology of ES cells and strong AP activity. Bar, 50mm. (e) Blastomeric cell culture from seabream at passage 6, showing smaller cells with ES-like morphology and intense AP staining, as well as larger cells apparently differentiated into fibroblastic-like type and without AP activity (arrowheads). Bar, 50mm. (e) Cells from the SaBE 1 culture at passage 12 showing a strong expression of the GFP gene marker, after being selected with the G418 drug. Bar, 50mm.
J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129
Fig. 1.
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J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129
Fig. 2. Chromosome number distribution of cells from SaBE 1 culture at passages 2, 8, 20 and 40. The n values (number of metaphases counted) corresponds to 21, 24, 26 and 30, respectively.
complish further tests of totipotency such as histochemical markers or telomerase activity, which proved very efficient in mouse [22] and chicken [5]. Additionally the in vitro differentiation into different cell types derived from all germ layers, will be induced. The final recognition as true ES cells would need however the in vivo proof of totipotency by injecting the cells into recipient embryos. The ability of these cells to be transfected is very important, either to introduce a desired mutation to be transmitted in a mendelian way, or as an in vivo gene marker, to check the contribution of ES cells to the chimeras. Cells were transfected with the pEGFP-N1 plasmid (Clontech Laboratories GmbH, Heidelberg, Germany), using a modification of the CaPO4 coprecipitation method [23]. The highest transfection efficiency was 1/4000 cells. The cells expressing GFP successfully responded to the positive selection by the drug G-418, after being kept for about 50 days under the selection conditions (Fig. 1(f)). Both the successful transformation of the SaBE1 cells with the pEGFP-N1 plasmid and its apparently efficient expression, represent important steps in the development process of ES-cells in S. aurata. They open the possibility for the homologous recombination process by using a suitable construction on one side and to check the fate of the ES-cells in the receptor embryo by means of the vital GFP marker, on the other. Actually genetic labeling with a GFP construct proved the most powerful approach for the in vivo screening chimeras of medaka [9].
Acknowledgements The authors acknowledge the Co. Cupimar, S.A. (San
Fernando, Ca´diz), for providing the eggs and the facilities. This work was supported by Grant BIO2-CT930430 from EU. References [1] Evans MJ, Kaufman MH. Nature 1981;292:154 – 6. [2] Martin GR. Proc Natl Acad Sci USA 1981;78:7634 – 8. [3] Bradley A, Evans M, Kaufman MH. Nature 1984;309:255– 6. [4] Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ. J Reprod Fertil 1991;43:255 – 60. [5] Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut J, Etches RJ. Development 1996;122:2339 – 48. [6] Collodi P, Kamei Y, Sharps A, Weber D, Barnes D. Mol Mar Biol Biotechnol 1992;1:257 – 65. [7] Sun L, Bradford CS, Ghosh C, Collodi P, Barnes DW. Mol Mar Biol Biotechnol 1995;4:193 – 9. [8] Hong Y, Winkler C, Schartl M. Mech Dev 1996;60:33–44. [9] Hong Y, Winkler C, Schartl M. Proc Natl Acad Sci USA 1998;95:3679 – 84. [10] Be´jar J, Hong Y, Alvarez MC. CIHEAM J Cah Options Mediterr 1998;34:131 – 9. [11] Lin S, Long W, Chen J, Hopkins N. Proc Natl Acad Sci USA 1992;89:4519 – 23. [12] Wakamatsu Y, Ozato K, Hashimoto H, Kinoshita M, Sakaguchi M, Iwamatsu T, Hyodo-Taguchi Y, Tomita H. Mol Mar Biol Biotechnol 1993;2:325 – 32. [13] Hong Y, Schartl M. Mol Mar Biol Biotechnol 1996;5:93– 104. [14] Flechon JE. In: Houbine LM, editor. Transgenic animals: Generation and use. Part II. Amsterdam: Harwood and Poubl, 1997:157 – 65 section F. [15] Wakamatsu Y, Ozato K, Sasado T. Mol Mar Biol Biotechnol 1994;3:185 – 91. [16] Alvarez MC, Otis J, Amores A, Guise K. J Fish Biol 1991;39:817 – 24. [17] Be´jar J, Borrego JJ, Alvarez MC. Aquaculture 1997;150:143– 53. [18] Sola L, Cataudella S. Boll Zool 1978;45:242.
J. Be´jar et al. / Genetic Analysis: Biomolecular Engineering 15 (1999) 125–129 [19] Longo L, Bygrave A, Grosveld FG, Pandolfi PP. Transgenic Res 1997;6:321 – 8. [20] Robertson EJ. In: Robertson EJ, editor. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: UK:IRL. Press, 1987:71 – 112.
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[21] Nichols J, Evans EP, Smith AG. Development 1990;110:1341–8. [22] Solter D, Knowles BB. Proc Natl Acad Sci USA 1978;75:5565– 9. [23] Friedenreich H, Schartl M. Nucl Acids Res 1990;18:3299– 305.