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10 11

12 13 14 15

16

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P. W. (1989) Bio/Technology 7, 143-145 Nilsson, K., Buzsaky, F. and Mosbach, K. (1986) Bio/Technology 4, 989-990 Griffiths, J. B. (1990) in Animal Cell Biotechnology (Spier, R. E. and Griffiths, J. B., eds), Vol. 4, pp. 147-165, Academic Press Cahn, F. (1990) Trends Biotechnol. 8, 131-136 Griffiths, J. B., Cameron, D. R. and Looby, D. (1987) Dev. Biol. Stand. 66, 331-338 Atkinson, B. and Mavituna, F. (1983) Biochemical Engineering and Biotechnology Handbook MacMillan Looby, D. and Griffiths, J. B. (1987) in Modern Approaches to Animal Cell Technology (Spier, R. E. and Griffiths, J. B., eds), pp. 342-352 Butterworth Brown, P. C., Figueroa, C., Costel]o, M. A. C., Oakley, R. and Maciukas, S. M. (1988) in Animal Cell Bioteehnology (Spier, R. E. and Griffiths, J. B., eds), Vol. 3, pp. 251-262

Academic Press 17 Whiteside, J. P. and Spier, R. E. (1981) Biotechnol. Bioeng. 23, 551-565 18 Burbidge, C. (1980) Dev. Biol. Stand. 46, 169-172 19 Looby, D. and Griffiths, J. B. (1988) Cytotechnology 1,439-446 20 Looby, D. and Griffiths, J. B. (1989) in Advances in Animal Cell Biology and Technology for Bioprocesses (Spier, R. E., Griffiths, ]. B., Stephenne, J. and Crooy, P., eds), pp. 336-344, Butterworth 21 Reiter, M. et al. Cytotechnology 3 (in press) 22 Young, M. W. and Dean, R. C. (1987) Bio/Technology 5,835-837 23 Arathoon, A. and Birch, J. (1986) Science 232, 1390-1395 24 Schott Glaswerke, Mainz Product Information No. 6196e 25 Payne, G. F., Shuler, M. L. and Brodelius, P. (1987) in Large Scale Cell Culture Technology (Lydersen, B. K., ed.), pp. 193-229, Hansen 26 Looby, D., Racher, A., Griffiths, J. B.

27

28

29

30

and Dowsett, A. B. (1990) in Physiology of Immobilised Cells (De Bont, J. A. M., Visser, J., Mattiasson, B. and Tramper, J., eds), pp. 255-264, Elsevier Runstadler, P. W. and Cernek, S. R. (1988) in Animal Cell Biotechnology (Spier, R. E. and Griffiths, J. B., eds), Vol. 3, pp. 305-320, Academic Press Mignot, G., Ganne, V., Faure, T. and van de Pol, H. (1989) in Proceedings of Cell Culture Engineering II Meeting Santa Barbara, USA Karkare, S. B., Phillips, P. G., Burke, D. H. and Dean, R. C. (1985) in Largescale Mammalian Cell Culture (Feder, J. and Tolbert, W. R., eds), pp. 127-150, Academic Press Bisping, B. and Rehm, H. J. (1986) Product Information No. 6196e, Schott Glaswerke, Mainz DENIS LOOBY AND BRYAN GRIFFITHS

Division of Biologics, PHLS CAMR, Porton Down, Salisbury, Wiltshire SP4 0JG, UK.

®®

Transgenic fish Thomas T. Chen and Dennis A. Powers A range of transgenic animal species have been generated using DNA microinjection, and application of this technique to fish is now showing some degree of success. Studies to optimize microinjection techniques specifically for use with fish, and to investigate possible alternative methods for mass culture, should lead to the commercial production of transgenic fish able to transmit desirable characteristics, such as enhanced growth or disease resistance, to their progeny. Animals into w h i c h heterologous genes have been artificially introd u c e d are t e r m e d transgenic. Since the early 1980s, transgenic C. elegans 1, Drosophila 2,3, sea u r c h i n s 4,5, frogs 6-9, laboratory mice (Refs 10, 11, see Ref. 12 for review), and farm

T. T. Chen is at the Center of Marine Biotechnology, University of Maryland, 600 East Lombard Street, Baltimore, MD 21202, USA and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, MD 21228, USA, and D. A. Powers is at the Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950, USA. ~) 1990, Elsevier Science Publishers Ltd (UK)

m a m m a l s s u c h as pigs, sheep and cows 13,14 have been p r o d u c e d by m i c r o i n j e c t i o n of heterologous DNA. In each of these cases, the DNA was injected into the p r o n u c l e i of fertilized eggs, and the injected embryos were t h e n i n c u b a t e d in vitro or imp l a n t e d into the uterus of a pseudopregnant female for s u b s e q u e n t d e v e l o p m e n t . These studies s h o w e d that m u l t i p l e copies of foreign genes integrated at r a n d o m locations into the g e n o m e of the transgenic animals in head-to-tail t a n d e m arrays. If a foreign gene was i n t r o d u c e d with a functional p r o m o t e r into the developing embryos, e x p r e s s i o n of that

0167 - 9430/90/$2.00

gene was e x p e c t e d in some of the transgenic individuals. In m a n y instances, the foreign genes were also t r a n s m i t t e d through the germ line to s u b s e q u e n t generations. T h e ability to generate and s t u d y transgenic animals is p r o v i d i n g n e w insights into m e c h a n i s m s of d e v e l o p m e n t and gene regulation, actions of oncogenes, a n d the intricate interactions w i t h i n the i m m u n e system. This t e c h n i q u e also offers the possibility of engineering animal m o d e l s for specific h u m a n genetic diseases and m a y be used for the p r o d u c t i o n of e c o n o m i c a l l y i m p o r t a n t proteins. Transgenic animals m a y s h o w altered p h e n o t y p e s . For instance, transgenic mice expressing h u m a n or rat growth h o r m o n e genes m a y possess elevated levels of growth h o r m o n e , and c o n s e q u e n t l y grow m u c h faster t h a n their control siblings 15. These results suggest it m a y be possible to i n t r o d u c e desirable genetic characteristics such as e n h a n c e d growth rates, disease resistance and cold resistance to comm e r c i a l l y i m p o r t a n t farm animals, replacing or c o m p l e m e n t i n g the conv e n t i o n a l genetic breeding approach. A t t e m p t s to p r o d u c e transgenic fish began only a few years ago, yet the results of this research effort are v e r y encouraging 16,~7. We r e v i e w the

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TIBTECH - AUGUST 1990 [Vol. 8]

remarkable progress made in the application of this technology to problems in fisheries science and basic biology.

Methods ofgene transfer Microinjection. Since 1985, heterologous genes introduced into fish by microinjection include the genes for human or rat growth hormone, chicken dalta-crystalline protein, E. coli beta-galactosidase, E. coli neomycin resistance gene, E. coli hygromycine resistance, winter flounder antifreeze protein, and the cDNA of rainbow trout growth hormone (Table 1). Direct microinjection of cloned DNA into the male pronuclei of fertilized eggs has proven to be the most successful and effective method of introducing foreign genes into the germ line of transgenic mice 1°,11, cows, pigs, sheep and rabbits 13,1~. Although the method requires specialized equipment and technical skill, it is conceptually straightforward, and any cloned DNA can be used. The technical aspects of the microinjection procedure for generating transgenic mammals and some important parameters for optimizing integration of foreign DNA have been reviewed extensively ~2,36,37. Pronucleus microinjection involves the following basic steps: • several hundred copies of linearized cloned genes are dissolved in a small volume (< 2 nl) and delivered into the nuclei of one-celled embryos by a glass micro-pipette; • the embryos are re-implanted into the uterus of the pseudopregnant females for further development; • DNA is extracted from the biopsy tissue of these presumptive transgenic animals and subjected to Southern blot hybridization analysis, using the radiolabelled heterologous gene as a probe; • P1 transgenic individuals raised for further studies.

are

The same microinjection technique has been used successfully to generate transgenic fish in many species such as common carp, catfish, goldfish, loach, medaka, salmon, Tilapia, rainbow trout, and zebrafish 16,18-35. Since pronuclei from most fish species studied to

~Table I S u m m a r y o f transgenic fish studies conducted by various laboratories, 1985-1990 Fish

Gene

Promoter

Ia

E

T

Year

+ +

+ +

1989 1989 1989 1987 1985 1988 1986 1986 1989 1989 1988 1989 1988 1988 1986,

Common carp Chinese carp Catfish Catfish Goldfish Goldfish Loach Medaka Medaka Medaka Salmon Salmon Salmon Tilapia Trout

rtGHcDNA hGH rtGHcDNA hGH hGH E. coli neo hGH c CR rtGHcDNA fLus E. coli[J-gal hGH fAFP fGH hGHcDNA

RSV mMT RSV mMT mMT SV40 mMT SV40 mMT fLuc mMT mMT fAFP mMT mMT

+ + + + + + + + + + + + + + +

Trout Trout Zebrafish

rGH cG E. coli hygro

mMT cG mMT

+ + +

+ + + + + + +

1988 1987 1989 1988

Ref, 18,19 18 20 21 22 23 24 25 26 27 28 29 30 31 32, 33 16 34 35

aAbbreviations: I, integration; E, expression; T, transmission

date cannot be easily visualized, the DNA is usually injected into the cytoplasm instead. Eggs and sperm from mature individuals are collected and placed into separate dry containers. Fertilization is initiated by adding water and sperm to eggs, with gentle stirring to facilitate the fertilization process. Egg shells are hardened in water for various periods of time, depending on the fish species. About 106 to 108 molecules of linearized DNA in a volume of 20 nl or less (Table 2) are microinjected into each egg within the first few hours after fertilization (i.e. between one-cell to four-cell stages). Following microinjection, eggs are incubated in appropriate hatching trays and dead embryos are removed daily. Since fish undergo external fertilization, the injected embryos do not require the complex manipulations essential in mammalian systems, such as in vitro culturing of embryos and transferring of embryos into foster mothers. A further advantage is that cytoplasmic injection in fish is less harmful to the embryos than injection into the nucleus (because the nuclei in fish cells are hard to locate), so the survival rate of injected fish embryos is much higher than that of mammalian embryos; depending on species, the survival

rate of injected fish embryos ranges from 35% to 80% (Table 2). Although the DNA is injected into the cytoplasm, the rates of DNA integration in transgenic fish are rather high (ranging from 10% to 70%). One exception to cytoplasmic injection is in the case of medaka, in which the pronuclei are visible so the foreign DNA can be injected into pronuclei a7,25. The tough chorion of the fertilized eggs of many fish species can make insertion of glass micro-needles difficult. This problem can be overcome in several ways. In rainbowtrout and salmon eggs, an opening is made by micro-surgery prior to insertion of the micro-needle 28,32. In Atlantic salmon and Tilapia, foreign DNA can be injected through the micropile, an opening for sperm penetration during fertilization 3°,31. While the chorion of zebrafish eggs can be removed manually with a pair of forceps 25, the chorion of goldfish and loach eggs is removed by trypsin digestion 22,24. Recently Oshiro et a]. 34 reported that hardening of rainb o w trout egg chorion could be prevented if fertilization took place in a solution containing 1 mM glutathione solution (pH 8.0). The problem of a tough chorion may in some cases be avoided by microinjection of the foreign DNA into unfertilized

TIBTECH - A U G U S T 1990 [Vol. 8]

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eggs, since the chorion of unfertilized eggs can be penetrated easily by a glass microneedle. Gene Transfer by Other Methods. Although cytoplasmic microinjection is a successful technique, it is a tedious and time-consuming procedure and unsuitable for mass gene transfer. Alternative methods therefore need to be developed. Unlike micro-injected DNA, retroviral genetic material is integrated into the genome of the infected cells very efficiently. Only a single copy of the proviral DNA is inserted at a given chromosomal site, and rearrangements of the host genome are not induced 38. Heterologous genes inserted into the viral genome can therefore be transferred to the host by viral infection. However, the lack of characterized species-specific retroviruses for fish has prevented the development of this method for gene transfer. Electroporation uses brief electrical pulses to permeabilize the cell membrane, thereby allowing entry of macromolecules, including DNA. This method for gene transfer has proved successful in bacteria, cultured mammalian cells and plant protoplasts (for review see Ref. 39). Despite numerous attempts by several laboratories, no success in the use of electroporation for transferring foreign genes into fish embryos has been reported. Liposomes prepared from phospholipids by the reverse phase evaporation method 4° have been used successfully as vehicles for delivering drugs and proteins into

mammalian cell cultures, and for encapsulating foreign DNA for transfection of mammalian cells or plant protoplasts 4~. While several groups, including our own, are attempting to introduce foreign DNA into fish embryos by this approach, there has been little success to date. Foreign DNA has also been introduced into rice or corn embryonic callus cells by high velocity microprojectiles (particle gun bombardment or biolistics) 42. In this method, foreign DNA is adsorbed onto 4 Fm spherical tungsten particles and transferred into plant cells by a particle gun. Since the particle can penetrate the plant cell wall, this method may be able to transfer foreign DNA through the intact chorion.

Transfer of growth hormone gene for growth enhancement Rainbow trout growth hormone (GH1) cDNA was expressed at a high level in E. coli 43. Application of the protein to yearling rainbow trout by intraperitoneal injection resulted in significant growth enhancement weight gain doubled following treatment with the recombinant GH for four weeks at i ~tg/g body weight/ week. The same growth-promoting effect was also observed in rainbow trout fry (two week old) receiving the recombinant growth hormone treatment via osmotic shock 43. Growth enhancement had also been reported with recombinant GH from other sources 43,44. While exogenous application of recombinant GH enhances somatic growth of cultured fish, the use of gene transfer technology to

produce transgenic fish expressing higher levels of GH is a more costeffective alternative. These fish not only produce the hormone endogenously, bypassing the production and delivery procedures, but they also pass on the enhanced growth characteristic to their progeny. In 1985, Zhu e t a ] . 22 reported successful transfer of human GH gene fused to a mouse metallothionein gene promoter into goldfish. The F1 offspring of these transgenic fish grew to twice the size of their non-transgenic siblings (Z. Zhu, pers. commun.), consistent with the growth enhancement effects of biosynthetic GH injected into rainbow trout 43 and salmon 44. Although Zhu et al. did not present compelling evidence for integration and expression of the foreign genes, a number of laboratories have successfully demonstrated that GH gene constructs can be transferred into eggs of a number of fish species and integrated into the fish's genomic DNA 17-21,23,25-35. Although a few groups have demonstrated expression of the foreign GH gene in transgenic fish 18-2°,29, at present, we are the only group to document that heterologous fish GH genes: (a) are transferred to the target fish species; (b) are integrated into the fish genome; (c) are genetically transmitted to the next (F1) generation; (d) are expressed in both P1 and F1 generations and (e) the expression of the foreign GH gene increases the growth rate of both the P1 and F~ generations of the transgenic

fish18-2o. In studies by Zhang et a]. 19, about

- - T a b l e 2,

Microinjection of foreign genes into fish eggs Fish Stage of Dealing species developwith ment chorion Carp, Catfish Trout Salmon Zebrafish

1-4 cells

-

Medaka

1-cell 2 steps 1-cell micropyle 1-4cells dechlorination oocyte -

Mouse

1-cell

-

Injection site

DNA form

Gene copies

Volume injected

cytoplasm

linear (5.2 kb)

10 6

20 nl

cytoplasm cytoplasm cytoplasm

linear linear (7.8 kb) linear (5.2 kb)

2×10 8 106 5×10 6

germinal circular vesicle (14.4 kb) male linear pronucleus 0.5-50

Survival (%) 40(hatching)

20 nl 77 (hatching) 2-3 nl 50(fry) 300 pl 16(fry)

0.5-1 ×104 10-20 pl 50 (embryo) 540

2 pl

19 (fetus)

Integration (%)

Ref.

10 (adult)

18-20

75 (embryo) 7 (fry) 5 (adult)

22,33 30 35

50 (embryo)

26

25 (fetus)

63

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--Fig. 1 106 molecules of a linearized recombinant plasmid containing the long terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and a trout GH1 cDNA were microinjected into the cytoplasm of one-cell, twocell, and four-cell common carp embryos. Genomic DNA samples extracted from the pectoral fin of individuals derived from these embryos were analysed by dot blot and Southern blot hybridization, using the LTR of RSV and trout GH1 cDNA as probes. The survival rate of the microinjected embryos at hatching was 35%, of which about 10% of the survivors were found to have stably integrated the pRSVLTR-rtGH1cDNA sequence. Furthermore, expression of trout GH polypeptide was also detected in many transgenic individuals. Although there was considerable variation in sizes of these P1 transgenic fish, they were on average 20% larger than their sibling controls (Fig. 1). Furthermore, a randomly selected fraction of the F1 progeny (which were derived from crosses between the P1 transgenic males and a non-transgenic female) inherited the foreign gene. Not only did those transgenic progeny grow faster than their nontransgenic siblings, they also grew much faster than their parents (Table 3). A gene transfer experiment involving a human GH gene construct gave similar results, with Chinese crucian carp F1 offspring which were substantially larger than the P1 mosaic parents being produced, but no significant differences were found when mirror carp were used (Ref. 18 and Z. Zhu, pers. commun./. Although the transfer, expression, and inheritance of a heterologous GH gene has been achieved, and some of the resulting transgenic fish grew considerably faster than their non-transgenic siblings, the generation of commercial strains of fastgrowing transgenic fish is still at the development stage. To achieve this goal, research in the following areas is required: (1) improvement of gene transfer efficiency; (2) identification of a suitable promoter to drive the expression of the foreign GH gene; (3) determination of physiological, nutritional, and environmental factors that will maximize the performance of transgenic individuals and (4) assessment of safety and environmental impact.

Transgenic carp. The top two larger individuals are mosaic P~ transgenic carp carrying and expressing the pRSVrtGH1 cDNA, while the bottom smaller individual is a P7 non-transgenic sibling.

Transfer of antifreeze protein genes for cold tolerance Fish are found in cold polar waters, warm tropical seas, and temperate waters where thermal conditions vary on a daily or seasonal basis. To adapt to these thermal conditions, they have developed sophisticated and efficient long term behavioural and reproductive strategies and physiological mechanisms. Some fish species in cold waters have evolved a novel set of proteins, the antifreeze proteins (AFP) that

prevent their blood freezing. De Vries (unpublished PhD thesis, University of Stanford, USA, 1969 and Ref. 45) found that expression of AFP genes in polar fish occurs all year round while temperate species, like winter flounder, express AFPs only in winter 47. These proteins allow the fish to live in water at a temperature of a b o u t - 2 ° C . Fish such as salmon and other salmonids that do not have the same or a similar adaptive strategy cannot survive at these temperatures.

T I B T E C H - AUGUST 1990 [Vol. 8] "

213

The AFPs of some Antarctic fish are composed of repeating units of Ala-Ala-Thr with a disaccharide, galactosyl-n-acetylgalactose amine, glycosidically linked to the threonine via a glycosidic bond 46,4s. On the other hand, the AFPs of the winter flounder are alanine-rich helical proteins that lack disaccharides 47. The mechanism by which these AFPs bind micro-ice crystals and thereby lower the freezing point of blood has been determined 49, as have the DNA sequences encoding these proteins in several fish species 5°-55. Recently, Huang e t al. 56 introduced the cloned winter flounder AFP genes into cell lines derived from rainbow trout, bluegill and salmon, and observed their expression by detecting AFP mRNA synthesis, indicating that AFP genes could possibly be used to produce cold resistant transgenic fish. Fletcher and his colleagues have recently attempted to introduce AFP genes of winter flounder into Atlantic salmon that lack AFP genes 3°. About 106 copies of a linearized plasmid containing AFP genes dissolved in 2 - 3 n l were microinjected into salmon eggs through the micropile. Out of 1800 injected eggs, about 80% survived to hatching. When analysis was carried out 8 months after hatching, the AFP gene

was detected in two out of 30 fingerlings. Although integration of the AFP gene into the salmon genome was established in this study, the expression of the gene in transgenic individuals was not detected. Recently, Fletcher and his colleagues 65 have obtained preliminary evidence showing the expression of winter flounder AFP gene in their transgenic salmon. However, the levels of AFP gene expression are still too low to provide protection against freezing. Transfer of antisense DNA sequence for disease resistance In any aquaculture operation, animal diseases account for a major loss of revenue and often make the difference between the success or failure of a particular commercial enterprise. The diagnosis and control of fish diseases caused by bacteria, fungi and viruses continues to present major technical and economic problems. Although antibiotics and chemotherapeutics can be used to control the spread of some fish pathogens efficiently, there are no commercially available antiviral drugs or vaccines for fish viral diseases. Therefore, the only effective solution in the industry has been the total destruction of virally infected fish. Infectious haematopoietic necro-

--Table 3

Mean weight, weight range and percent inheritance at 90 days of progeny derived from transgenic fish 131L and 94r 19

Character Sample size % inheritance Mean weight (g) Standard deviation Coefficient of variation Weight range (g) % difference

P r o g e n y o f p a r e n t 131L

P r o g e n y o f p a r e n t 94r

Transgenic Non-transgenic

Transgenic Non-transgenic

31 32.3 120.6 a 17.4

65 99.3 b 14.7

11 42.0 206 45.2

15 147 48

14.6

14.9

21.9

32.6

95-173 c 20.8 e

65-129 40.1 f

115-283 d -

67-228 -

aN = 28 bN = 38 c32% of transgenic progeny were larger than largest control d46% of transgenic progeny were larger than largest control eTransgenic progeny were larger than non-transgeric p r o g e n y at P < 0.05 ~ r a n s g e n i c p r o g e n y were larger than non-transgenic p r o g e n y at P < 0.001

sis virus (IHNV) was identified as a pathogen causing massive death in sockeye salmon in the state of Washington in 1953. Since then, the disease has spread from Alaska to California and as far west as Japan, and has threatened both natural and cultured populations of salmon and trout in recent years. Because of the economic impact of IHN and related diseases, efforts are being made to develop vaccines against IHNV. Recently, Leong and co-workers 57-6° have cloned and sequenced the IHNV nucleocapsid gene and a cDNA of the IHNV surface glycoprotein. By expressing the cDNA sequence of this glycoprotein gene in a baculovirus expression vector, they have succeeded in producing large quantities of biosynthetic vaccine, which has been shown to be effective in treating rainbow trout infected with IHNV. In addition to vaccines for IHNV and other related viral diseases, a recombinant vaccine has been developed for the treatment of a bacterial pathogen belonging to the genus V i b r i o 61. There are two alternative strategies to the anti-viral vaccination approach, both of which involve the generation of immune fish, but neither has yet been tried. One involves the introduction of DNA coupled to an appropriate promoter to ensure expression of an antisense viral RNA sequence, such that viral infection is blocked by preventing replication of the virus. This strategy has proved effective in avian retroviral systems 62. Alternatively, the transfer of a viral envelope protein gene into an organism might provide protection against the virus. Adequate concentrations of the viral envelope protein would need to be produced by the host to compete for the receptor binding .sites for the virus on the cell surface. Future prospects Although there has been substantial progress in the generation of transgenic fish since 1985, further research is required in order to take full advantage of fish as experimental systems and to develop trausgenic fish for commercial aquaculture purposes. Development of efficient methods for introducing genes into the large numbers of eggs produced would make fish a particularly attractive

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TIBTECH- AUGUST 1990 [Vol. 8]

Acknowledgements

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