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iii GENE TRANSFER IN FISH Eric M. Hallerman I, Kevin S. Guisel, 5, Anne R. Kapuscinski 2, Perry Hackett, jr.3, 5, and Anthony J. Faras4, 5 B. Departments of iAnimal Science, 2Fisheries and Wildlife, 3Genetics Cell Biology, 4Microbiology, and 5Institute for Human Genetics, University of Minnesota, St. Paul, MN 55108. and Although conventional breeding programs in fish parallel those in chicken, unconventional techniques not available in chicken breeding, such as intersmecific hybridization (Schwartz, 1981), ploidy manipulation and gynogenesis (Thorgaard and Allen, 1987), are used in breeding of fishes. Within the last five years, another unconventional means of genetic manipulation, gene transfer, has become available to animal breeders. Techniques utilized for gene transfer in fish are quite different from those utilized in chicken. In this report, the methods and status of gene transfer in fish are reviewed, with many aspects of progress in the field exemplified with results from our own work in the Minnesota Transgenic Fish Group. Demonstration of dramatic growth in mice bearing introduced growth factor genes (Palmiter et al., 1982; _Hammer et al., 1985) created widespread interest in the possibility of producing agricultural livestock exhibiting enhanced productivity through the action of introduced genetic constructs. Because of the possible value to aquaculture, gene transfer in fish has attracted a relatively large research effort. At least 23 laboratories (Table i), nine in the U.S. and at least 14 Ln other countries, are involved in gene transfer in fish. The broad scope of the research effort has led to rapid developaent of protocols for gene transfer in fish, and the successful production of transgenic fish of at least 13 species (Table 2). METHODOI/3GY OF GENE TRANSFER iN FISH Successful gene transfers in fish have been accomplished through microinjection of newly-fertilized fish eggs. Gene transfer protocols for particular fishes are variations upon a generalized series of steps, which is ex_nplified below by our protocol for production of transgenic goldfish. Induction of spawning. Goldfish can be brought to spontaneous ovulation through appropriate manipulation of photoperiod and temperature, with injections of carp pituitary extract if necessary (Stacy et al., 1979). Gamete collection. tively ripe individuals toward the vent. Gametes are readily collected from reproducby gentle manual stroking of the abdcmen 112 Fertilization. Eggs and semen are mixed in a petri dish. Activation of the gametes and fertilization are effected by addition of water. Dechorionation. Removal of the chorion facilitates effective microinjection into the animal pole of the egg. Enzymatic digestion offers a practical means for rapid, en masse dechorionation of fish eggs. Dechorionation of goldfish eggs is carried out by a brief incubation of the water-hardened eggs in 0.25% trypsin solution (Yoon et al., in press). Enzymatic dechorionation procedures have not, however, proven practical for all species, including certain species of acf_aculture interest (Hallerman et al., 1988). Lack of a dechorionation protocol for eggs of rainbow trout led to use of a broken pipette to make a small hole in the chorion of the egg prior to microinjection (Chourrout et al., 1986). Other groups (Fletcher et al., 1988; Indiq and Moav, 1988) have circumvented the problem of injecting through the chorion by microinjecting through the micropyle of the egg. For other species, such as walleye and northern pike, it has proven practical to microinject eggs imnobilized by their natural stickiness onto agarose petri dishes. .... • Microinjection. Inserted prcmoter/coding region sequences are removed from plasmid vectors, purified, and resuspended in TE (i0 mM tris, 1 mM EDTA; pH 7.4). The DNA solution is loaded into borosilicare glass needles (tip diameter approximately 2 microns). Microinjection is performed using either a simple system driven by oil pressurized by a syringe (Brinkman, 06-90-24) or using a so dhisticated system driven by nitrogen pressurized by a unit regulating pressure and injection time (Medical Systems Corp., PLI-100). In either case, the aim is to inject DNA into the animal pole of the egg prior to first cleavage. In a notable exception to the approach described, Ozato et al. (1986) microinjected mature ooc_es dissected from med_ ovaries, thus introducing the novel genetic construct prior to fertilization. Incubation. Goldfish eggs are incubated through hatch and yolk sac resorption in petri dishes. Embryos and larvae of other species such as northern pike, walleye, and rainbow trout, which require incubation in cool, moving water, are incubated in trays specifically designed for the purpose (Heath Techna Corp. ). Growout. Goldfish are grown out in aquaria until roughly 5 cm in length, and in large (approximately 300 i) troughs thereafter. Northern pike, walleye, and rainbow trout are grown out in cylindrical fiberglass tanks (approximately 500 1 volt,he). The n_nber of fish which can be grown out under laboratory confin_nent frequently limits the size of gene transfer experiments. 113 SUCCESS OF TRANSFER AND EXPRESSION Transfer efficiencies. range from 3% (Br_n et al., communication), with 20% of transformant. OF INTRODUCED CCNSTRUCI_ Transfer efficiencies reported for fish 1988) to over 70% (Z. Zhu, personal injected embryos more typically proving Expression of introduced constructs. Most gene transfer experiments to date have aimed at production of faster-growing fish through introduction of growth-prc_oting genetic constructs. Zhu et al. (1985) first reported transfer of the human growth _hormone gene into goldfish, and have since reported expression, germ-line tr2m_nission, and growth rate enhancement among transgenic goldfish _nd loach (Maclean et al., 1987b). Transfers of various grc_*_h hormone genes have subsequently been documented for several fish species (Table 2), and accelerated growth of transgenic carp (Zhang et al., 1988) and northern pike (Schneider et al., in press) have beech reported. Representing a contrasting approach to genetic improv_r__nt of fishes, the antifreeze protein of winter flounder has be=_n cloned and transferred into Atlantic salmon (Fletcher et al., 1988 ). Ln winter flounder, this gene encodes a protein which depresses _ freezing temperature of sera in the fish, allowing them to survive in supercooled _aters. Results of early experiments indicated that levels of antifreeze gene expression were too low to provide protection to transgenic individuals. If sufficient levels of expression are ultimately achieved, the transferred antifreeze protein ge_ne could expand the range of sites in which sea-cage aquaculture of salmon could be carried out. Among gene transfers in fish to date, the levels of expression of introduced genes have not generally been high. Many groups have introduced genetic constructs containing the murine metallot_hionein promoter modelled after those used in the classic studies of transgenic mice. However, prcmoters from simian virus 40 (SV40) and Rous sarc(_na virus (RSV) have produced higher levels of expressian than the murine metallothionein pramoter (Stuart et al., 1988 ). In avian systems, the RSV promoter seems to give rise to expression kn muscle and connective tissues, the desired sites for expression of growthprcmoting constructs. In northern pike (Schneider et al., Ln press ) and goldfish (Yoon et al., in press), roughly one-third of transg_nic fish showed expression of constructs bearing the RSV prcmoter. F[/fURE ADVAN___S IN GENE TRANSFER L-N FISH Beyond the inclusion of additional species, future progress in gene transfer will ce_ter upon utilization of new expression vectors. Production of improved expression vectors will involve testing and practical utilization of new regulatory and protein-_ncoding genetic elements. Rec2/latory elements. The success of any gene transfer is dependant upon the level, tissue-specificity, and ontoge_netic stagespecificity of expression of the novel gene product, factors which are 114 determined by the function of the regulatory elements in the expression vector. The challenge is to find and utilize regulatory elements giving rise to patterns of gene expression appropriate to the purposes of the experiment. Hence, expression of growth-prcmoting factors would be optimized by use of a promoter which is highly active in muscle tissue throughout the juvenile stage, but w_ich deactivates upon sexual maturity. In contrast, synthesis of antifreeze peptides would optimally be restricted to cold seasons of the year, perhaps with regulation mediated in response to levels of serotonin in the serum of the fish. The success of gene transfers aimed at elimination of nutritional requirements attributable to, say, lack of a particular enzyme, would depend upon constitutive levels of gene expression in all tissue types. Future advances in gene transfer in fish are thus highly dependant upon discovery, characterization, and utilization of new prcmoter or enhancer elements. As development of such elements is and will remain a focus of research activity, it is not surprizing that groups involved in gene transfer are generally reluctant to discuss experiments in progress. _ _ . :- Structural genes of econanic interest. The second functional component of an expression vector is the coding sequence for the novel gene product. Anticipated progress inproduction of new expression vectors will center upon incorporation of additional growth prcmoting genes. The search for genes which prcraote gr(m/ch of fish has intensified. Growth hormone genes of rainbow trout (Agellon _nd Chen, 1986), gilthead sea bream (Cavari et al., 1988), chum salmon (Sakine et al., 1985), chinook salmon (C. Hew, personal ccrm_unication), coho salmon (Nicoll et al., 1987; Gonzalez-Villasenor et al., 1988), yellow±ail (Watahiki et al., 1988 ), tuna (Sato et al., 1988 ), and red sea bream (Mcmota et al., 1988) have been cloned. In higher vertebrates, growth is regulated by a cascade of growth factors, with hypothalamic gro%ech hormone releasing factor triggering the release of pituitary growth hormone, in turn triggering the production of the insulin-like growth factor I in the liver, which ultimately binds to receptors in somatic cells to prcmote growth. The degree to which this endocrine cascade is paralleled in fish is currently urf_nown. For purposes of investigating this question, efforts will be devoted to cloning such piscine genes using mams_lian seque_nces as probes, and to transfer of mammalian gro_dn factor genes into fish, followed by observations for enhanced gr(_ch. It is indeed an open question as to what other broad types of structural genes might be attractive candidates for gene transfer in fish. Chum salmon prolactin has been cloned (Song et al., 1988), and represents the start of efforts to clone other piscine peptide hormone genes. Targetted integration. Because introduced constructs are integrated into the host genome at random locations, there is a considerable probability that such integration events will result in insertional deactivation of a host gene. Furthermore, gene expression is to some_ degree dependant upon the location of a gene within the gencme. Hence, direction of integration of a novel construct to specific sites within the genome is important not only in gene therapy, 115 but in a broad range of applications. Direction of integration in gene therapy is approached through inclusion of promoter and coding elements between flanking sequences homologous to the desired site of gencmic integration; it is hoped that the cell's own DNA repair mechanisms will splice the introduced construct within the defective gene. Parallel gene transfer experiments have be__n initiated in fish, using flanking sequences corresponding to sequences repeated within the piscine genQ_e. MASS GENE TRANSFER Microinjection is a tedious and time-consuming procedure. In our laboratory, the collective efforts of six or more people are required in order to microinject i0,000 fish eggs per day. The labor required to produce the large number of transgenic fish frcm which to select an optimal gene transfer event is thus a factor limiting the success of gene transfer experiments. Hence, there is interest in developing mass gene transfer technologies for use in fish so that a large number of eggs may be treated at one time, with use of a subsequent selection step to distinguish transgenic individuals. At least t_hree approaches to mass gene transfer are being investigated: Electroporation. Electroporation is the utilization of short electrical pulses to permeabilize the cell membrane, thereby gaining entry of vector DNA into the cytosol, and ultimately the gencme, of target cells. The utilization of electroporation for gene transfer, which has proven successful in bacteria, cultured mamnalian cells, and plant protoplasts (Shigekawa and Dower, 1988), has also been attempted upon fertilized, dechorionated fish eggs. Although we .have defined conditions that do not kill goldfish eggs, we have yet to produce transformant individuals using this technique (Hallerman et al., in press ). Lipofection. Liposome-mediated gene transfer (Felgner et al., 1987) is based on the encapsulation of vector DNA into a synthetic phospholipid bilayer, which fuses with the cell membrane of the target cell, introducing the foreign DNA into the cell. For purposes of gene transfer in fish, the target cells would be dechorionated fish eggs. While several groups, including ours, are attempting this mode of gene transfer, no success has been reported. Adsorption of vector DNA onto sperm. In preliminary experiments in our laboratory, upwards of 10% of radiolabelled pla_nid DNA (suspended in s_nen extender) mixed with fish se_en has remained adsorbed to sperm cells through three cycles of washing in semen extender and centrifugation. In fish species lacking t_be acroscme reaction, the entire sperm enters the egg during fertilization. These observations lead us to believe that DNA adsorbed onto sperm in this manner might transform embryos so fertilized. This procedure would have £he potential to effect gene transfer upon .many eggs with relatively little effort. 116 Selectable markers. Transfer efficiencies in mass transfer pro- tocols are expected to be low, and so it is preferable that a selectable marker (perhaps necn_ycin resistance; Southern and Berg, 1982) or a gene that gives rise to a phenotypic marker be used. Such selection regimes for fish embryos are under development (Yoon et al., in press ). USE OF FISH AS MODEL SYSTEMS FOR VERTEBRATE GENE EXPRESSION In studies of vertebrate gene expression using gene transfer methodology, mouse is the most widely used model system. However, ccmparisen of their respective life histories reveals features which render fish more suited for gene transfer experiments: --the fecundity of fish is much higher, increasing the number of eggs available for microinjection, and ultimately, the number of transgenic individuals produced, --fertilization and incubation of fish eggs is external, circumventing the need for in vitro fertilization of mouse eggs and reimplantation into a surrogate mother, --fish eggs are relatively large, facilitating handling, --transformation frequencies of microinjected fish eggs equal or exceed those of mouse eggs, and --the action of regulatory sequences in fishes parallels those in higher vertebrates. We thus believe that piscine systems will be more widely used as model systems in future studies of vertebrate gene expression. Goldfish. Because of wide availability, ease of culture, and ease of inducing spawning, goldfish may became_ an important model system. The drawback of goldfish is the long (cne year) generation time relative to mouse or certain other fishes, lengthening the duration of studies depending upon germ-line transmissions of novel genetic constructs. Zebrafish. With a four-month generation time, zebrafish are an attractive model system for studies requiring germ-line transmission. Additionally, maps tracing the fates of particular cells in the early embryonic stages have been developed (Kimmel and Warga, 1988), providing the basis for studies of ontogenetic gene expression and regulation. USE OF TRANSGENIC FISH IN AQUACULTURE Applications of gene transfer technology may have major impacts on aquaculture breeding programs. The successful production of growth enhanced reca_binant fish strains might have wide application and large econcmic returns. Although rapid progress has been achieved in production of transgenic fishes exhibiting favorably altered phenotypes, several factors will delay the use of transgenic fish in aquaculture. 117 Development of improved sterilization techniques. Production of sterile fish is desirable for proprietary protection of transgenic bro0dstock and as a means of minimizing the environmental impacts consequent to escape of transgenic individuals frcm an aquaculture facility. Three techniques might be used to produce functional sterility in fish: polyploidization (Thorgaard and Allen, 1987), interspecific hybridization (Schwartz, 1981), and hormonal treatment (Donaldson _nd Hunter, 1983). Protocols combining two or more of these approaches might be developed, as sane individuals escape sterilization using any one of these methods alone. Breeding constraints. The transgenic individuals produced in gene transfer experiments are not th_nselves intended as production animals, but as progenitors of new genetic lines. Several generations of breeding will be required to bring the frequency of novel genes to high frequencies and to carry out performance evaluations before transgenic lines can be utilized in conventional aquaculture breeding programs (Figure 1 ). The time constraint introduced by breeding considerations is important, as the generation times in aquaculture species (e.g., three years in rainbow trout and channel catfish) are long. Legal constraints. Both the natural environmental conditions required for attainment of sexual maturity in certain species and the normal culture conditions required for identification of high performance lines dictate the need for outdoor containment during development of transgenic lines. Controlled environmental testing of organisms altered through recombinant DNA technology is a controversial element of public policy. Development of animals bearing introduced DNA constructs is regulated under the Coordinated Framework for the Regulation of Biotechnology (Office of Science and Technology Policy, 1984; 1985; 1986). Provisional policy guidelines regulating outdoor testing of transgenic animals have been prcmulgated by the Agricultural Biotechnology Research Advisory Committee of the U.S. Department of Agriculture. No outdoor testing of transgenic animals of any kind has yet been carried out, although a permit has recently been granted for outdoor release of transgenic carp (Ezzell, 1989). Development of public policies regarding the ultimate practical utilization of transgenic animals is proving controversial, with many interest groups voicing their concerns. Policies regarding field testing, and ultimately, distribution and final use of transgenic fishes should reflect a fundam__ntal difference bet_en aquaculture fishes and traditional agriculture _nimals. Transgenic fishes can beccme feral, giving rise to environmental impacts directly through their altered phenotypes or by reproducing within natural populations of conspecifics. Practical utilization of transgenic fishes in the U.S. may thus be delayed by develo_Qment of field testing data and analysis of such data within risk asses_nent models. 118 ACKNOWLEDGEMENTS We would like to thank Program Chairman Ramakrishna Reddy for his help in arranging this presentation. Our funding support came frcm the Minnesota Agricultural Experiment Station, the Legislative Commission for Minnesota Resources, and fr(xn Minnesota Sea Grant. _TUREC_D Agellon, L.B., and T.T. Chen. 1986. Rainbow Molecular cloning of cDNA and expression DNA 5: 463-467. trout growth hormone: in Escherichia coli. Brem, G., G. 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Biological effects of human growth hormone gene microinjected into the fertilized eggs of loach Misgurnus anguillicaudatus (Cantor). Kexue Tongbao 31: 988-990. 123 TABLE I. Scope of world effort on gene transfer Country United in fish. Species States: Auburn/Johns Rainbow trout, carp, catfish Channel catfish channel rainbow pike, Idaho Goldfish, zebrafish, trout, northern walleye Rainbow trout Oregon Purdue Zebrafish Medaka Southern Illinois Washington State Tilapia Rainbow Louisiana Hopkins/Maryland State Minnesota Abroad--in Canada sp. trout, salmon operation: (2 ) Rainbow trout, salmon Atlantic England France Rainbow trout Rainbow trout, salmon Germany Ireland Tilapia sp., Xiphophorus Atlantic salmon Israel (3) Tilapia sp., Gilthead bream Japan Norway (3) People's Republic Abroad--status India, Atlantic Medaka Atlantic salmon Chinese carps, loach of China unknown: Indonesia, Hungary, Malaysia, Thailand, U.S.S.R. sea sp. Table 2. Gene transfers in fish. Species Atlantic Salmon Channel catfish Common carp Construct 1 124 Status 2 Refe re nce mMT-bGal I,E mMT-rGH I Rokkones et al. 1988 AFP-AFP I Fletcher et al. 1988 mMT-hGH I Dunham et al. 1987 - I Joyce et al. 1988 mMT-hGH I Maclean et al. 1987b mMT-sGH McEvoy et al. 1988 Joyce et al. 1988 RSV-tGH I,E,R,G mMT-hGH I mMT-hGH I,G Maclean et al. 1987b RSV-neo mMT-hGH I,T I,R Yoon et al. in press Zhu et al. 1986 mMT-hGH I,R Maclean et al. 1987b Medaka mMT-cCRY I,E Ozato et al. 1986 Mud carp mMT-hGH I Northern pike Rainbow trout RSV--bGH SV40-hGH I,E I mMT-rGH I Goldfish Loach Zhang et al. 1988 Zhu et al., 1985 Maclean et al. 1987b Schneider et al. in press Chourrout et al. 1986 Maclean et al. 1987a mMT-rGH mMT-rGH Guyomard et al. 1988 Penman et al. 1988 mMT-rGH Rokkones et al. 1988 Silver crucian mMT-hGH I Maclean et al. 1987b Tilapia nilotica mMT-hGH I Brem et al. 1988 SV40-CAT I,E Indiq and Moav 1988 Wuchang fish mMT-hGH I Maclean et al. 1987b Zebra fish SV40-hyg I Stuart et al. 1988 RSV-CAT I,E,G Stuart et al. in rev. 1 Thegeneticconstructsintroducedinto fish are referredto as fusiongenes,comprisedof a promoter sequencewhichregulatesexpressionof the gene,and a structuralsequencewhich encodesthe protein productof the geneof interest. Abbreviationsfor promotersequences:mMT,mousemetallothionein; AFP,antifreezeprotein;RSV,Roussarcomavirus;SV40,simianvirus40. Abbreviationsfor structural genes:bGal,beta-galactosidase;AFP, antifreezeprotein;hGH, humangro_'_hhormone;tGH, rainbow troutgrowthhormone;sGH,salmongrowthhormone;neo,neomycinresistance,CAT,chloramphenicol transacetylase;bGH,bovinegrowthhormone,cCRY,chickencrystalline;rGH, rat growthhormone,and hyg, hygromycinresistance. 2 Statusdesignations:I, genomicintegration;T, transcription;E, expressionof protein;G, germline transmission;R, rapidgrowth. 125 FIGURE i. Development of transgenic lines of fish for utilization conventional aquaculture breeding programs. Original Generation N20% carry new gene, mostly as 'mosaics' _->Non-Transgenics k'_Not Transformed P Half of of_pring hemizygous for new I First Generation gene . More Inbreeding Performance Crossbreeding Aquaculture Non-Tr.a.nsg Testing with Existing Lines enics in Germ Line in 126 Question: D. Harwood Are the fish oocytes microinjected with intact plasmids restriction fragments containing only the promoter and structural gene sequences? Response: or E. Hallerman We have miroinjected intact plasmid, linearized inserted promoter/structural gene elements into Although our data are presently incomplete, the is as follows: - Highest levels of transient expression lowing introduction of intact plasmid. - Genomic form. integration seems - Levels of stable expression seem highest when only the introduced. more frequent are plasmid, and fish eggs. picture that observed using fol- linearlized of the introduced construct promoter/coding elements are Thus, I would advocate microinjec_ion of the promoter/coding elements only for the purposes of most gene transfer experiments, when genomic integration and stable expression are desired. Please note also that we inject the whatever form, into fertilized ova, expression vector, in rather than oocytes.