Cell Differentiation and Development, Elsevier Scientific Publishers Ireland.
CELDIF
29 (1990) 123-128 Ltd.
123
00640
Electroporation
as a new technique for producing transgenic fish
Koji Inoue, Shinya Yamashita,
Jun-ichiro Hata, Shoko Kabeno, and Takao Fujita
Sachiko Asada, Eizo Nagahisa
Central Research Laboratory, Nippon Suisan Kaisha, Hachioji, Tokyo, Japan (Accepted
27 October
1989)
A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 ps, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the Fl offspring assayed. In F2 offspring obtained by mating transgenic Fl fish, il%% were transgenic. Gene transfer; Transgenic fish; Electroporation;
Microinjection;
Introduction
Transgenic animals are a powerful tool for in vivo studies of the regulation and function of genes and have been produced in various species such as fruit flies (Rubin and Spradling, 1982; Spradling and Rubin, 1982) frogs (Rusconi and Schaffner, 1981; Etkin, 1982) nematodes (Stinchcomb et al., 1985), mice (Gordon et al., 1980; for a review see Palmiter and Brinster, 1986) and fish (for a review see Maclean et al., 1987; Ozato et al., 1989). Transgenic animals have been produced mostly by microinjection of foreign DNA into the nucleus or cytoplasm of fertilized eggs. In the case of fish, microinjection into eggs involves certain difficulties. The nucleus cannot be
Correspondence address: S. Yamashita, Central Laboratory, Nippon Suisan Kaisha, Kitano-machi, Tokyo 192, Japan. 0922-3371/90/.$03.50
0 1990 Elsevier Scientific
Research Hachioji,
Publishers
Ireland,
Medaka; Growth hormone
found optically in the fertilized eggs of fish. The cytoplasm of fish eggs in many species is covered by a tough and/or opaque chorion which prevents insertion of glass micropipettes. To overcome these difficulties, special methods have been developed for microinjection into fish eggs, e.g., injection into oocyte nuclei (Ozato et al., 1986) penetration of micropipettes through the chorion by two-step injection in salmonids (Chourrout et al., 1986; McEvoy et al., 1988) injection through the micropile in tilapia (Brem et al., 1988) and Atlantic salmon (Fletcher et al., 1988) and dechorionization in goldfish (Zhu et al., 1986) loach (Zhu et al., 1985) and zebrafish (Stuart et al., 1988). Thus, the microinjection technique in fish eggs is complicated and requires a great deal of skill, and obstructs progress in transgenic research on fish. To avoid the difficulty accompanying microinjection, we have attempted to apply electroporation (Neumann et al., 1982; Potter et al., 1984) which is often used for gene transfer into culture Ltd.
124
cells, to the production of transgenic fish. We report here on the successful introduction of a plasmid including rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, into a small teleost, medaka (Oryzius iutipes), by electroporation and germ-line transmission of the transgene to the offspring.
Materials and Methods Structure and preparation of the recombinant plasmid The recombinant plasmid pMV-GH (Fig. 1) contains the metal responsive region of mouse metallothionein I (mMT-I) promoter, rainbow trout growth hormone (rtGH) cDNA and the polyadenylation sequence of SV40. After linearization with EcoRI, this plasmid was dissolved in mannitol buffer containing 0.25 M mannitol, 0.1 mM CaCl,, 0.1 mM MgCl,, 0.2 mM Tris-HCl (pH 7.5) at the concentration of 100 pg/ml for electroporation or in Dulbecco’s phosphate-buffered saline at the concentration of 10 pg/ml for microinjection. Egg collection The orange-red strain of medaka (25-35 mm in total length) was used for egg collection. About 15
5.2kb
I
rtGH
lkb
fish were maintained each in a 30-l aquarium at 26 o C under an artificially controlled photoperiod (14 h light and 10 h dark). In this condition, they spawn eggs every day at the beginning of the light period (Yamamoto, 1975). To harmonize the time of fertilization, males and females were kept separate during the dark period and mated at the beginning of the light period. Spawning usually occurs within 5 min after mating. Spawned egg clusters were taken from the abdomens of females about 20 min after mating. The attachment filament was cut off with fine scissors. Electroporation Isolated eggs were put between the electrodes of a transfection chamber, Shimadzu FTC-03 (electrode distance, 2 mm), filled with 800 ~1 of the DNA solution (100 pg/ml). With this chamber, about 120 medaka eggs could be treated at a time. Before first cleavage, electric pulses were applied by a gene transfer equipment (Shimadzu GTE-l) under the following condition: pulse height, 750 V/cm; pulse interval, 1 s; pulse length 50 ps; pulse number, 5. Treated eggs were rinsed in distilled water several times and incubated separately in 96-well plastic cell wells filled with distilled water until hatching at 26 o C. Microinjection Microinjection was performed as previously described (Ozato et al., 1986; Inoue et al., 1989). About 30 pl of the DNA solution (10 pg/ml) was microinjected into each nucleus of the oocytes obtained from the female body cavity by laparotomy at 9 h before the beginning of the light period. Microinjected oocytes were incubated until ovulation in Earle’s 199 medium supplemented with 2% bovine serum albumin, 17.8 mM NaHCO,, 25 mg/l penicillin G and 15 mg/l streptomycin sulfate, and inseminated. Fertilized eggs were incubated at 26 o C in 96-well plastic cell wells filled with distilled water until hatching.
I
Fig. 1. Structure of the recombinant plasmid, pMV-GH. The solid bar represents the metal-responsive promoter sequence of mouse metallothionein I (mMT-I). The open bar represents rainbow trout growth hormone (rtGH) cDNA. The hatched bar represents the polyadenylation sequence of simian virus 40 (SV40). The lines indicates vector sequence of pUC19.
Slot blot and Southern analysis Genomic DNA was extracted from whole bodies of hatchlings or tail fin pieces of adult fish. After incubation in 300 ~1 of lytic buffer containing 50 mM Tris-HCI (pH 7.5) 10 mM EDTA,
0.5% SDS, 500 pg/ml ProteinaseK at 55°C for 4 h, samples were extracted once with an equal volume of phenol and then twice with phenol: chloroform : isoamyl alcohol (24 : 24 : 1). The aqueous phase was then precipitated with ethanol and dissolved in TE (10 mM Tris, 1 mM EDTA). The DNA sample for slot blotting was denatured in 0.2 M NaOH for 10 min, neutralized by adding an equal volume of 2 M NH,OAc, and immediately fixed onto a nylon membrane. The DNA sample for Southern blotting was digested with EcoRI and Hind111 or Z-%1, electrophoresed on 0.7% agarose gels, and transferred onto a nylon membrane. Membranes were hybridized to the EcoRI-StuI fragment of rtGH cDNA labelled by random priming with [32P]dCTP. The StuI site of rtGH cDNA corresponds to the junction of rtGH cDNA and the SV40 sequence in pMV-GH and was lost in the process of constructing the plasmid. Then filters were exposed to X-ray film.
Results Gene transfer by electroporation Of 3109 fertilized eggs treated with pulses, about 10% died immediately from damage to the yolk sac (Fig. 2). Of the surviving embryos, 783 (25%) hatched out at 8-20 days after fertilization. To examine the efficiency of gene transfer, Southern blot hybridization was performed on 180 hatchlings, and 7 (4% of hatchlings analyzed) were identified as transgenic. A typical result of Southern blotting is shown in Fig. 3. In Fig. 3, the correct size of the transgene is 2.6 kb, because genomic DNA was digested with EcoRI and Hind111 and probed with rtGH cDNA. The copy number of the transgene in each positive individual varied from over 100 (Fig. 3, lane 6) to 1 (data not shown). In an individual (Fig. 3, lane l), one major band at 5.2 kb and two minor bands at 2.6 kb and 1.5 kb were observed. The size of the major band corresponds to that of the whole pMV-GH. This may be caused by integration of several copies of transgene in a head-to-tail manner after deletion of the EcoRI site used to linearize the plasmid. The minor band at 1.5 kb may be an end of the transgene. Only the 2.6-kb band main-
Fig. 2. Early embryos of medaka at 5 h after application of electric pulses. Several embryos were damaged by electric pulses (arrows). Bar = 10 mm.
tamed the correct size. No band was from untreated fish (data not shown).
detected
Gene transfer by microinjection To compare the gene transfer efficiency of electroporation with that of microinjection, pMV-GH was also introduced by microinjection into oocytes.
1234567
5.2kb 2.6kb 1.5kb
Fig. 3. Southern blot analysis treated with electric pulses. tracted from whole larvae Hind111 and hybridized
of medaka fry hatched from eggs About 1.5 pg genomic DNA exwas digested with EcoRI and with the rtGH cDNA probe.
126
1
Of 198 injected oocytes, 102 (52%) were fertilized and 70 (35%) hatched out. By Southern analysis, 7 (27%) of 26 assayed hatchlings were identified as positive. The Southern blotting pattern was basically identical to that in electroporation (data not shown). Germ-line transmission of transgene To establish transgenic lines bearing pMV-GH, 180 fry hatched from the eggs treated with electric pulses were maintained and 35 matured within 3 months. By slot blot hybridization on DNA extracted from the tail fin of each individual, 2 fish were identified as transgenic (data not shown). One was male and the other was female. Since the female died soon after maturation, we failed to obtain offspring from this fish. From the male, about 100 eggs were obtained by mating a normal female. Southern analysis was performed on 17 Fl offspring. Surprisingly, the transgene was detected in all the individuals analyzed (Fig. 4). In these Fl offspring, the copy number of transgene was estimated at 1 copy per cell. No band was detected in control fish (Fig. 4, lane 10). The transgenic Fl offspring also matured within 3 months. By mating a pair of Fl offspring, we could obtain F2 offspring. Southern blot analysis of the F2 offspring (Fig. 5) showed that 21 out of 24 (88%) F2 offspring assayed inherited the transgene. The copy number of the transgene in posi-
12
3
4
5
6
7
8
91011
2.4kb
Fig. 4. Southern blot analysis of the Fl offspring obtained from the transgenic founder male and a normal female. About 5 pg of the PstI digest of genomic DNA extracted from Fl fish (lanes 1-9) and a control fish (lane 10) was hybridized with the rtGH cDNA probe. The plasmid pMV-GH digested with PsrI was also shown in lane 12 as the positive control.
2
3
4
5
6
Fig. 5. Southern blot analysis of F2 offspring obtained by mating a pair of transgenic Fl offspring. About 5 pg of the PstI digest of genomic DNA extracted from F2 fish (lanes 1-5) and a control fish (lane 6) was hybridized with the rtGH cDNA probe.
tive offspring (Fig. 5, lanes l-4) was estimated at approximately 1 or 2 per cell, although it could not be determined exactly. In Fig. 5 results on a negative individual and a normal fish were also shown in lanes 5 and 6, respectively. Thus, the transgenes were passed on to F2 offspring in a basically Mendelian fashion.
Discussion
Several methods have been reported for gene transfer into animals. For example, microinjection of DNA, infection with recombinant retroviruses and embryonic stem cell- or teratocarcinoma-cellmediated transfer are now available in mice (Palmiter and Brinster, 1986). In this study, we used electroporation as a new method for transfer of a foreign plasmid into a small freshwater fish, medaka. Electroporation has often been used for gene transfer into culture cells but not into embryos. This is the first successful application of electroporation for the production of transgenic animals. In fish, gene transfer is usually achieved by microinjection into the cytoplasm of fertilized eggs. However, microinjection into fish eggs is sometimes difficult because the chorion prevents insertion of micropipettes in many species. Electro-
127
poration makes gene transfer easier and the method used in this study is very simple. Foreign genes could be transferred merely by applying pulses to eggs in a DNA solution. Unlike microinjection no skilled technique is required for this method. A defect of this method is the low efficiency of gene transfer. The rate of transgenic individuals in hatchlings was only 48, far lower than the 27% rate in oocyte microinjection. However, this defect may be overcome by the fact that a large number of eggs can be treated at a time by electroporation. Fish generally spawn enormous numbers of eggs at one spawning. At this point, electroporation seems especially suited to gene transfer in fish. For practical use, selection markers to distinguish transgenic individuals from many nontransgenic ones, such as resistance to antibiotics, should be devised. One of the probable causes of the low efficiency in gene transfer is that the plasmid DNA could not come into contact with the cell membrane because of the presence of the chorion and perivitelline space. Yamaha et al. (1988) introduced exotic reagents into dechorionized goldfish eggs. Methods for dechorionization have already been established in goldfish (Yamaha et al., 1986; Zhu et al., 1985), loach (Zhu et al., 1986), medaka (Iwamatsu, 1983) and zebrafish (Stuart et al., 1988). It is possible that dechorionization increases the efficiency of gene transfer in this method. In this study, inheritance of the transgene through the germ line was demonstrated. This implies that the transgene was stably integrated into host chromosomes. Though germ-line transmission of the transgene is essential for the study of transgenic animals, it has never been demonstrated in fish except the zebrafish (Stuart et al. 1988). In the present study, the inheritance of the transgene by all the Fl offspring was unexpected. This may indicate that the transgene was integrated independently into both sets of the chromosomes although this remains to be proved. Expression of the transgene was not examined in this study. It will be examined in the near future on transgenic offspring which are now growing.
Acknowledgement We wish to express our thanks to Dr. Kenjiro Ozato for a critical reading of the manuscript.
References Brem, G., B. Brenig, G. Hiilstgen-Schwark and E.-L. Winnacker: Gene transfer in tilapia (Oreochromis niloricur). Aquaculture 68, 209-219 (1988). Chourrout, D., R. Guyomard and L.M. Houdebine: High efficiency gene transfer in rainbow trout (Salmo guirdneri) by microinjection into egg cytoplasm. Aquaculture 51, 143-150 (1986). Etkin, L.D.: Analysis of mechanisms involved in gene regulation and cell differentiation by microinjection of purified genes and somatic cell nuclei into amphibian oocytes and eggs. Differentation 21, 149-159 (1982). Fletcher, G.L., M.A. Shears, M.J. King, P.L. Davis and C.L. Hew: Evidence for antifreeze protein gene transfer in Atlantic salmon (S&no snlar). Can. J. Fish. Aquat. Sci. 45, 352-357 (1988). Gordon, J.W., G.A. Scangos, D.J. Plotkin, J.A. Barbosa and F.H. Ruddle: Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77,7380-7384 (1980). Inoue, K., K. Ozato, H. Kondoh, T. Iwamatsu, Y. Wakamatsu, T. Fujita and T.S. Okada: Stage-dependent expression of the chicken &crystallin gene in transgenic fish embryos. Cell Differ. Dev. 27, 57-68 (1989). Iwamatsu, T.: A new technique for dechorionization and observations on the development of the naked egg in Oryrias latipes. J. Exp. Zool. 228, 83-89 (1983). McEvoy, T., M. Stack, B. Keane, T. Barry, J. Sreenan and F. Gannon: The expression of a foreign gene in salmon embryos. Aquaculture 68, 27-37 (1988). Maclean, N., D. Penman and Z. Zhu: Introduction of novel genes into fish. Bio/Technology 5, 257-261 (1987). Neumann, E., M. Schaefer-Ridder, Y. Wang and P.H. Hofschneider: Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841-845 (1982). Ozato, K., H. Kondoh, H. Inohara, T. Iwamatsu, Y. Wakamatsu and T.S. Okada: Production of transgenic fish: introduction and expression of chicken d-crystallin gene in medaka embryos. Cell Differ. Dev. 19, 237-244 (1986). Ozato, K., K. Inoue and Y. Wakamatsu: Transgenic fish: biological and technical problems. Zool. Sci. 6, 445-457 (1989). Palmiter, R.D. and R.L. Brinster: Germ-line transformation of mice. Am. Rev. Genet. 20, 465-499 (1986). Potter, H., L. Weir and P. Lader: Enhancer-dependent expression of human II immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984).
128 Rubin, G.M. and A.C. Spradling: Genetic transformation of Drosophila with transposable element vectors. Science 218, 348-353 (1982). Rusconi, S. and W. Schaffner: Transformation of frog embryos with a rabbit-globin gene. Proc. Natl. Acad. Sci. USA 78, 5051-5055 (1981). Spradling, A.C. and G.M. Rubin: Transformation of cloned P-elements into Drosophila germ-line chromosomes. Science 218, 341-347 (1982). Stinchcomb, D.T., J.E. Shaw, S.H. Carr and D. Hirsh: Extrachromosomal DNA transformation of Cuenorhubditis elegant. Mol. Cell. Biol. 5, 3484-3496 (1985). Stuart, G.W., J.V. McMurray and M. Westerfield: Replication, integration and stable transmission of foreign sequences injected into early zebrafish embryos. Development 103, 403-412 (1988).
Yamaha, E., K. Usui, H. Onozato and K. Hamada: A method for dechorionization in goldfish, Curamiur auratus. Nippon Suisan Gakkaishi 52, 1929-1934 (1986). Yamaha, E., M. Matsuoka and F. Yamazaki: Introduction of exotic reagents into denuded eggs of goldfish and crucian carp by electroporation. Nippon Suisan Gakkaishi 54, 2043 (1988). Yamamoto, T.: Medaka (Killifish) Biology and Strains (Keigaku Publ. Co., Tokyo) (1975). Zhu, Z., G. Li, L. He and S. Chen: Novel gene transfer into the fertilized eggs of goldfish (Carasrir4.r aurutw L. 1758). 2. Angew. Ichthyol. 1, 31-34 (1985). Zhu, 2.. K. Zu, G. Li, Y. Xie and L. He: Biological effects of human growth hormone gene microinjected into the fertilized eggs of loach Misgurnus angdicaudatus (Cantor). Kexue Tongbao 31, 988-990 (1986).
CELDIF
29 (1990) 123-128 Ltd.
123
00640
Electroporation
as a new technique for producing transgenic fish
Koji Inoue, Shinya Yamashita,
Jun-ichiro Hata, Shoko Kabeno, and Takao Fujita
Sachiko Asada, Eizo Nagahisa
Central Research Laboratory, Nippon Suisan Kaisha, Hachioji, Tokyo, Japan (Accepted
27 October
1989)
A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 ps, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the Fl offspring assayed. In F2 offspring obtained by mating transgenic Fl fish, il%% were transgenic. Gene transfer; Transgenic fish; Electroporation;
Microinjection;
Introduction
Transgenic animals are a powerful tool for in vivo studies of the regulation and function of genes and have been produced in various species such as fruit flies (Rubin and Spradling, 1982; Spradling and Rubin, 1982) frogs (Rusconi and Schaffner, 1981; Etkin, 1982) nematodes (Stinchcomb et al., 1985), mice (Gordon et al., 1980; for a review see Palmiter and Brinster, 1986) and fish (for a review see Maclean et al., 1987; Ozato et al., 1989). Transgenic animals have been produced mostly by microinjection of foreign DNA into the nucleus or cytoplasm of fertilized eggs. In the case of fish, microinjection into eggs involves certain difficulties. The nucleus cannot be
Correspondence address: S. Yamashita, Central Laboratory, Nippon Suisan Kaisha, Kitano-machi, Tokyo 192, Japan. 0922-3371/90/.$03.50
0 1990 Elsevier Scientific
Research Hachioji,
Publishers
Ireland,
Medaka; Growth hormone
found optically in the fertilized eggs of fish. The cytoplasm of fish eggs in many species is covered by a tough and/or opaque chorion which prevents insertion of glass micropipettes. To overcome these difficulties, special methods have been developed for microinjection into fish eggs, e.g., injection into oocyte nuclei (Ozato et al., 1986) penetration of micropipettes through the chorion by two-step injection in salmonids (Chourrout et al., 1986; McEvoy et al., 1988) injection through the micropile in tilapia (Brem et al., 1988) and Atlantic salmon (Fletcher et al., 1988) and dechorionization in goldfish (Zhu et al., 1986) loach (Zhu et al., 1985) and zebrafish (Stuart et al., 1988). Thus, the microinjection technique in fish eggs is complicated and requires a great deal of skill, and obstructs progress in transgenic research on fish. To avoid the difficulty accompanying microinjection, we have attempted to apply electroporation (Neumann et al., 1982; Potter et al., 1984) which is often used for gene transfer into culture Ltd.
124
cells, to the production of transgenic fish. We report here on the successful introduction of a plasmid including rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, into a small teleost, medaka (Oryzius iutipes), by electroporation and germ-line transmission of the transgene to the offspring.
Materials and Methods Structure and preparation of the recombinant plasmid The recombinant plasmid pMV-GH (Fig. 1) contains the metal responsive region of mouse metallothionein I (mMT-I) promoter, rainbow trout growth hormone (rtGH) cDNA and the polyadenylation sequence of SV40. After linearization with EcoRI, this plasmid was dissolved in mannitol buffer containing 0.25 M mannitol, 0.1 mM CaCl,, 0.1 mM MgCl,, 0.2 mM Tris-HCl (pH 7.5) at the concentration of 100 pg/ml for electroporation or in Dulbecco’s phosphate-buffered saline at the concentration of 10 pg/ml for microinjection. Egg collection The orange-red strain of medaka (25-35 mm in total length) was used for egg collection. About 15
5.2kb
I
rtGH
lkb
fish were maintained each in a 30-l aquarium at 26 o C under an artificially controlled photoperiod (14 h light and 10 h dark). In this condition, they spawn eggs every day at the beginning of the light period (Yamamoto, 1975). To harmonize the time of fertilization, males and females were kept separate during the dark period and mated at the beginning of the light period. Spawning usually occurs within 5 min after mating. Spawned egg clusters were taken from the abdomens of females about 20 min after mating. The attachment filament was cut off with fine scissors. Electroporation Isolated eggs were put between the electrodes of a transfection chamber, Shimadzu FTC-03 (electrode distance, 2 mm), filled with 800 ~1 of the DNA solution (100 pg/ml). With this chamber, about 120 medaka eggs could be treated at a time. Before first cleavage, electric pulses were applied by a gene transfer equipment (Shimadzu GTE-l) under the following condition: pulse height, 750 V/cm; pulse interval, 1 s; pulse length 50 ps; pulse number, 5. Treated eggs were rinsed in distilled water several times and incubated separately in 96-well plastic cell wells filled with distilled water until hatching at 26 o C. Microinjection Microinjection was performed as previously described (Ozato et al., 1986; Inoue et al., 1989). About 30 pl of the DNA solution (10 pg/ml) was microinjected into each nucleus of the oocytes obtained from the female body cavity by laparotomy at 9 h before the beginning of the light period. Microinjected oocytes were incubated until ovulation in Earle’s 199 medium supplemented with 2% bovine serum albumin, 17.8 mM NaHCO,, 25 mg/l penicillin G and 15 mg/l streptomycin sulfate, and inseminated. Fertilized eggs were incubated at 26 o C in 96-well plastic cell wells filled with distilled water until hatching.
I
Fig. 1. Structure of the recombinant plasmid, pMV-GH. The solid bar represents the metal-responsive promoter sequence of mouse metallothionein I (mMT-I). The open bar represents rainbow trout growth hormone (rtGH) cDNA. The hatched bar represents the polyadenylation sequence of simian virus 40 (SV40). The lines indicates vector sequence of pUC19.
Slot blot and Southern analysis Genomic DNA was extracted from whole bodies of hatchlings or tail fin pieces of adult fish. After incubation in 300 ~1 of lytic buffer containing 50 mM Tris-HCI (pH 7.5) 10 mM EDTA,
0.5% SDS, 500 pg/ml ProteinaseK at 55°C for 4 h, samples were extracted once with an equal volume of phenol and then twice with phenol: chloroform : isoamyl alcohol (24 : 24 : 1). The aqueous phase was then precipitated with ethanol and dissolved in TE (10 mM Tris, 1 mM EDTA). The DNA sample for slot blotting was denatured in 0.2 M NaOH for 10 min, neutralized by adding an equal volume of 2 M NH,OAc, and immediately fixed onto a nylon membrane. The DNA sample for Southern blotting was digested with EcoRI and Hind111 or Z-%1, electrophoresed on 0.7% agarose gels, and transferred onto a nylon membrane. Membranes were hybridized to the EcoRI-StuI fragment of rtGH cDNA labelled by random priming with [32P]dCTP. The StuI site of rtGH cDNA corresponds to the junction of rtGH cDNA and the SV40 sequence in pMV-GH and was lost in the process of constructing the plasmid. Then filters were exposed to X-ray film.
Results Gene transfer by electroporation Of 3109 fertilized eggs treated with pulses, about 10% died immediately from damage to the yolk sac (Fig. 2). Of the surviving embryos, 783 (25%) hatched out at 8-20 days after fertilization. To examine the efficiency of gene transfer, Southern blot hybridization was performed on 180 hatchlings, and 7 (4% of hatchlings analyzed) were identified as transgenic. A typical result of Southern blotting is shown in Fig. 3. In Fig. 3, the correct size of the transgene is 2.6 kb, because genomic DNA was digested with EcoRI and Hind111 and probed with rtGH cDNA. The copy number of the transgene in each positive individual varied from over 100 (Fig. 3, lane 6) to 1 (data not shown). In an individual (Fig. 3, lane l), one major band at 5.2 kb and two minor bands at 2.6 kb and 1.5 kb were observed. The size of the major band corresponds to that of the whole pMV-GH. This may be caused by integration of several copies of transgene in a head-to-tail manner after deletion of the EcoRI site used to linearize the plasmid. The minor band at 1.5 kb may be an end of the transgene. Only the 2.6-kb band main-
Fig. 2. Early embryos of medaka at 5 h after application of electric pulses. Several embryos were damaged by electric pulses (arrows). Bar = 10 mm.
tamed the correct size. No band was from untreated fish (data not shown).
detected
Gene transfer by microinjection To compare the gene transfer efficiency of electroporation with that of microinjection, pMV-GH was also introduced by microinjection into oocytes.
1234567
5.2kb 2.6kb 1.5kb
Fig. 3. Southern blot analysis treated with electric pulses. tracted from whole larvae Hind111 and hybridized
of medaka fry hatched from eggs About 1.5 pg genomic DNA exwas digested with EcoRI and with the rtGH cDNA probe.
126
1
Of 198 injected oocytes, 102 (52%) were fertilized and 70 (35%) hatched out. By Southern analysis, 7 (27%) of 26 assayed hatchlings were identified as positive. The Southern blotting pattern was basically identical to that in electroporation (data not shown). Germ-line transmission of transgene To establish transgenic lines bearing pMV-GH, 180 fry hatched from the eggs treated with electric pulses were maintained and 35 matured within 3 months. By slot blot hybridization on DNA extracted from the tail fin of each individual, 2 fish were identified as transgenic (data not shown). One was male and the other was female. Since the female died soon after maturation, we failed to obtain offspring from this fish. From the male, about 100 eggs were obtained by mating a normal female. Southern analysis was performed on 17 Fl offspring. Surprisingly, the transgene was detected in all the individuals analyzed (Fig. 4). In these Fl offspring, the copy number of transgene was estimated at 1 copy per cell. No band was detected in control fish (Fig. 4, lane 10). The transgenic Fl offspring also matured within 3 months. By mating a pair of Fl offspring, we could obtain F2 offspring. Southern blot analysis of the F2 offspring (Fig. 5) showed that 21 out of 24 (88%) F2 offspring assayed inherited the transgene. The copy number of the transgene in posi-
12
3
4
5
6
7
8
91011
2.4kb
Fig. 4. Southern blot analysis of the Fl offspring obtained from the transgenic founder male and a normal female. About 5 pg of the PstI digest of genomic DNA extracted from Fl fish (lanes 1-9) and a control fish (lane 10) was hybridized with the rtGH cDNA probe. The plasmid pMV-GH digested with PsrI was also shown in lane 12 as the positive control.
2
3
4
5
6
Fig. 5. Southern blot analysis of F2 offspring obtained by mating a pair of transgenic Fl offspring. About 5 pg of the PstI digest of genomic DNA extracted from F2 fish (lanes 1-5) and a control fish (lane 6) was hybridized with the rtGH cDNA probe.
tive offspring (Fig. 5, lanes l-4) was estimated at approximately 1 or 2 per cell, although it could not be determined exactly. In Fig. 5 results on a negative individual and a normal fish were also shown in lanes 5 and 6, respectively. Thus, the transgenes were passed on to F2 offspring in a basically Mendelian fashion.
Discussion
Several methods have been reported for gene transfer into animals. For example, microinjection of DNA, infection with recombinant retroviruses and embryonic stem cell- or teratocarcinoma-cellmediated transfer are now available in mice (Palmiter and Brinster, 1986). In this study, we used electroporation as a new method for transfer of a foreign plasmid into a small freshwater fish, medaka. Electroporation has often been used for gene transfer into culture cells but not into embryos. This is the first successful application of electroporation for the production of transgenic animals. In fish, gene transfer is usually achieved by microinjection into the cytoplasm of fertilized eggs. However, microinjection into fish eggs is sometimes difficult because the chorion prevents insertion of micropipettes in many species. Electro-
127
poration makes gene transfer easier and the method used in this study is very simple. Foreign genes could be transferred merely by applying pulses to eggs in a DNA solution. Unlike microinjection no skilled technique is required for this method. A defect of this method is the low efficiency of gene transfer. The rate of transgenic individuals in hatchlings was only 48, far lower than the 27% rate in oocyte microinjection. However, this defect may be overcome by the fact that a large number of eggs can be treated at a time by electroporation. Fish generally spawn enormous numbers of eggs at one spawning. At this point, electroporation seems especially suited to gene transfer in fish. For practical use, selection markers to distinguish transgenic individuals from many nontransgenic ones, such as resistance to antibiotics, should be devised. One of the probable causes of the low efficiency in gene transfer is that the plasmid DNA could not come into contact with the cell membrane because of the presence of the chorion and perivitelline space. Yamaha et al. (1988) introduced exotic reagents into dechorionized goldfish eggs. Methods for dechorionization have already been established in goldfish (Yamaha et al., 1986; Zhu et al., 1985), loach (Zhu et al., 1986), medaka (Iwamatsu, 1983) and zebrafish (Stuart et al., 1988). It is possible that dechorionization increases the efficiency of gene transfer in this method. In this study, inheritance of the transgene through the germ line was demonstrated. This implies that the transgene was stably integrated into host chromosomes. Though germ-line transmission of the transgene is essential for the study of transgenic animals, it has never been demonstrated in fish except the zebrafish (Stuart et al. 1988). In the present study, the inheritance of the transgene by all the Fl offspring was unexpected. This may indicate that the transgene was integrated independently into both sets of the chromosomes although this remains to be proved. Expression of the transgene was not examined in this study. It will be examined in the near future on transgenic offspring which are now growing.
Acknowledgement We wish to express our thanks to Dr. Kenjiro Ozato for a critical reading of the manuscript.
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