Download iii GENE TRANSFER IN FISH Eric M. Hallerman I, Kevin S. Guisel, 5

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. Brenig, G. Horstgen-Schwark,
and E.-L. Winnacker.
1988.
Gene transfer in tilapia (Oreochrcmis niloticus).
Aquaculture
68: 209-219.
Cavari,
B., B. Funkenstein,
T.T.
Chert, and D.A.
P(m_rs.
1988.
Cloning the growth hormone gene frcm the sea bream Spsrus aurata.
Third Intl. Conf. Genet. Aquacult., Trondheim, Norway, 20-24 June,
1988 (Abstract).
Chourrout,
D., R. Guycrnard, and L.M. Houdebine.
1986.
High efficiency gene transfer in rainbow trout (Salmo gairdneri Rich. ) by
microinjection
into egg cytoplasm.
Aquaculture
51: 1443-1150.
Donaldson, E.M., and G.A. Hunter.
1983.
Induced final maturation,
ovulation, and spermiation in cultured fish.
In: Fish
Physiology, Vol. 9B, pp.
405-435.
Ed. by Hoar, W.S., D.J.
Randall, and E.M. Donaldson.
Academic Press, Ne_ York.
Dunham, R.A., J. Eash, J. Askins, and T.M. Towns.
the metallothionein-human
hormone fusion gene
fish.
Trans. Am. Fish. Soc. 116: 87-91.
Ezzell,
C.
1989.
New carp park ahead.
Nature
1987.
Transfer of
into channel cat-
338:
366.
Felgner, P.L., T.R. Gadek, M. Holm, R. Rcman, H.W. Chan, M. Wenz,
J.P. Northrup, G.M. Ringold, and M. Danielsen.
1987.
Lipofection:
A highly efficient, lipid mediatedDNA
transfection
procedure.
Proc. Natl. Acad. Sci., U.S.A. 84: 7413-7417.
Fletcher, G.L., M.A. Shears, M.J. King, P.L. Davies, and C.L. Clew.
1988.
Evidence for antifreeze protein gene transfer in Atlantic
salmon (Salmo salar).
Can. J. Fish. Aquat. Sci. 45: 352-257.
Gonzalez-Villasenor,
L.I., P. Zhang, T.T. Chert, and D.A. P(m_rs.
1988.
Molecular cloning and sequencing of coho salmon growth
hormone cDNA.
Gene 65: 239-245.
119
Guy(m_ard, R., D. Chourrout,
and L.M. Houdebine.
1988.
Gene
transfer
by microinjection
into fertilized trout eggs:
Efficient integration and germ line transmission.
Third. Intl. Conf. Genet.
Aquacult., Trondheim, Norway, 20-24 June, 1988 (Abstract).
Hallerman, E.M., A.J. Faras, P.B. Hackett, A.R. Kapuscinski, and K.S.
Guise.
Attempted introduction of a novel gene into goldfish
t.hrough electroporation.
J. Cell. Biochem. 13B: in press
(Abstract).
Hallerman, E., J.F. Schneider, M.L. Gross, A.J. Faras, P.B. Hackett,
K.S. Guise, and A.R. Kapuscinski.
1988.
Enzymatic dechorionation of goldfish, walleye, and northern pike eggs.
Trans. Amar.
Fish. Soc. 117: 456-460.
Indiq, F.E., and B. Moav.
1988.
A prokaryotic gene is expressed in
fish cells and ._ersists in tilapia e_bryos and adults following
microinjection.
Pages 221-225 in Y. Zohar and B. Breton, eds.
Reproduction in Fish:
Basic and---applied aspects of endocrinology
and genetics.
INRA Press, Paris.
Joyce, C., M. Hayat, and R.A. Dunham.
1988.
Survival of channel catfish and ccrm_n carp embryos microinjected
with DNA at various
developmental stages.
Third Intl. Conf. Genet. Aquacult.,
Trondheim, Norway, 20-24 June, 1988 (._stract).
Ham_er, R.E., R.L. Brinster, M.G. Rosenfeld, R.M. Evans, and K.E.
Mayo.
1985a. Expression of human growth hormone-releasing
factor
in transgenic mice results in increased somatic growth.
Nature
315: 413-416.
Kimmel, C.B., and R.M. Warga.
1988.
Cell lineage and developmental
potential of cells in the zebrafish embryo.
Trends Genet. 4:
68-74.
Macle_n, N., D.
genes into
Selection,
Verlag, H.
Penman, and S. Talwar.
1987a.
Introduction of novel
the rainbow trout.
Pages 325-323 in K. Tiews, ed.
Hybridization,
and Genetic Engineeri---ngin Aquaculture.
Heenemann GmbH, Berlin.
Maclean, N., D. Pemman, and Z. Zhu.
genes into fish.
Bio/Technology
1987b.
Introduction
5: 257-261.
of novel
McEvoy, T., M. Stack, B. Keane, T. Barry, J. Sreenan, and F. Gannon.
1988. The expression of a foreign gene in salmon embryos.
Aquaculture 68: 27-37.
Mcmota, H., R. Kosugi, H. Hiramatsu, H. Ohgai, A. Hara, and H.
Ishioka.
1988. Nucleotide sequence of cDNA encoding the
pregrowth hormone of red sea bream (Pagrus ma'olg_r). Nuc. Acids
Res. 16: 3107.
120
Nicoll, C.S., S.S. Steiny, D.S. King, R.S. Nichioka, G.L. Mayer, N.L.
Ederhardt, J.D. Baxter, M.K. Yamanaka, J.A. Miller, J.J.
Seilhamer, J.W. Schilling, and L.K. Johnson.
1987.
The primary
structure of coho salmon growth hormone and its cDNA.
Gen. Comp.
Endocrinol.
68: 387-399.
Office of Science and Technology Policy.
1984.
Proposal for a coordinated framework for regulation of biotechnology.
Fed. Reg. 49:
50856-50907, December 31, 1984.
Office of Science and Technology Policy.
1985.
Coordinated framework
for the regulation of biotechnology;
Establishment
of the
biotechnology
science coordinating committee.
Fed. Reg. 50:
47174-47195, Nov_1_ber 14, 1985.
Office of Science and Technology
Policy.
1986.
Coordinated
framework
for regulation of biotechnology.
Fed. Reg. 51: 23301-23350, June
26, 1986.
Ozato, K., H. Kondoh, H. Inohara, T. Iwamatsu, Y. Wakamatsu, and T.S.
Koada.
1986.
Production of transgenic fish:
Introduction and
expression of chicken crystallin gene in medaka embryos.
Cell
Diff. 19:
237-244.
Palmiter, R.D., R.L. Brinster, R.E. Hammer, M.E. Trumbauer, M.G.
Rosenfeld, N.C. Brinberg, and R.M. Evans.
1982.
Dramatic growth
of mice that develop from eggs microinjected
with
metallothionein-growth
hormone fusion genes.
Nature 300:
611-615.
Penman, D.J., A.J. Beeching, S. Penn, and N. Maclean.
1988.
Factors
affecting the integration of microinjected
DNA into the rainbow
trout g_ncme.
Third Intl. Conf. Genet. Aquacult., Trondheim,
Norway, 20-24 June 1988 (.Abstract).
Rckkones, E., P. Alestrom, H. Skjervold, and K.M. Gautvik.
Expression of human growth hormone gene in fertilized
Atlantic salmon and rainbcw trout.
Third Intl. Conf.
Aquacult.,
Trondheim,
Norway,
20-24 June
1988
1988.
eggs from
Genet.
(Abstract).
Sato, N., K. Watanabe, K. Murata, M. Sakaguchi, Y. Kariya, S., Kimura,
M. Nonaka, and A. Kimura.
1988. Molecular cloning and
nucleotide sequence of tuna growth hormone cDNA.
Biochim.
Biophys. Acta 949: 35-42.
Schneider, J.F., E.M. Hallerman, S.J. Yoon, L. He, M.L. Gross, Z. Liu,
A.J. Faras, P.B. Hackett, A.R. Kapuscinski,
and K.S. Guise.
Transfer of the bovine growth hormone gene into northern pike,
Esox lucius.
J. Cell. Biochem. 13B: in press (Abstract).
121
Schwartz, F.J.
1981. World literature to fish hybrids with an analysis by family, species, and hybrid:
Suppl_nent I. NOAA
Technical Report hlMFS SSRF-750.
Environmental
Science_
Information Center (D822), NOAA, 609 Executive Blvd., Rockville,
MD.
Sekine, S., T. Mizukumi, T. Nishi, Y. Kuwana, A. Saito, M. Sato, S.
Itoh, and H. Kawauchi.
1985.
Cloning and expression of cDNA for
salmon growth hormone in Escherichia
coli.
Proc. Natl. Acad.
Sci., U.S.A. 82: 4306-4310.
Shigekawa, K., and W.J. Dower.
1988.
Electroporation
of eukaryotes
and prokaryotes : A general approach to the introduction of
macrcmolecules
into cells.
Biotechniques
6: 742-751.
Song, S., K.-Y. Trinh, C.L. Hew, S.-J. Hwang, S. Belkhode, and D.I.
Idler.
1988.
Molecular cloning and expression of salmon prolactin cDNA.
Eur. J. Bioch_n. 172: 279-285.
Southern, P.J., and P. Berg.
1982.
Transformation
of mamnalian cells
to antibiotic resistance with a bacterial gene under control of
SV40 early region promoter.
J. Mol. Appl. C__net. i: 327-341.
Stacy, N.E., A.F. Cook, and R.E. Peter.
1979.
Spontaneous and
gonadotrophin-induced
ovulation in the goldfish, Carassius
auratus L. Effects of external factors.
J. Fish. Biol. 15:
349-361.
Stuart, G.W., J.V. McMurray, and M. Westerfield.
1988. Replication,
integration, and stable germ-line transmission of foreign sequences into early zebrafish embryos.
Development
103: 403-412.
Stuart, G.W., J.R. Vielkind, J.V. McMurray, and M. Westerfield.
Germline transmission and expression of a r_inant
chloramphenicol
acetytransferase
gene in transgenic zebrafish.
In review.
Thorgaard, G.H., and S.K. Allen, Jr.
1987.
Chromoscme manipolation
and markers in fishery management.
Pages 319-331 in N. Ryman and
F. Utter, eds.
Population Genetics and Fishery Management.
University of Washington
Press, Seattle.
Watahiki, M., M. Tanaka, N. Masuda, M. Yamakawa, Y. Yoneda, and K.
Nakashima.
1988.
cDNA cloning and primary structure of
yellowtail (Seriola quinqueradiata)
pregrowth hormone.
Gen.
Ccmp. Endocrinol. 70: 401-406.
Yoon, S.J., E.M. Hallerman, M. Gross, Z. Liu, J.F. Schneider, A.J.
Faras, A.R. Kapuscinski, and K.S. Guise.
Transfer of the gene
for neomycin resistance into goldfish, Carassius auratus.
Aquaculture, in press.
122
Zhang, P., M. Hayat, C. Joyce, C.M. Lin, L.J. Gcnzalez-Villasenor, R.
Dunham, T.T. Chen, and D.A. Powers. 1988. Gene transfer,
expression, and inheritance of f_RSV-trout-GH-cDNAin fish. First
Intl. Symp. Marine Molec. Biotech. 9-11 Oct., 1988, Baltimore,
MD (Abstract).
Zhu, Z., G. Liu, L. He, and S. Chen. 1985. Novel gene transfer into
the fertilized eggs of goldfish (Carassius auratus L. 1758). Z.
Angew. Ichthyol. i: 31-34.
Zhu, Z., K. Xu, G. Li, Y. Xie, and L. He. 1986. 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.