1
Transgenic fish. The future
of fish with novel genes
Filip Volckaert
Geneticist
Frans Ollevier
Biologist
1 Introduction
Making transgeniclinesofvertebratesrepresentsa
rather new
technology. While transgenicfishhavebeenintroduced
inthe
laboratory since the n i d eighties, it has also becolne a reality in
aquaculture since the early nineties. There are many reasons why
people want to create genetically modified organisms (GMO) and
there are many strategies to nlake "fish with novel genes". As such
it has generated highexpectationsinSociety
and directed the
also
raised concerns about
interests of researchers, but has
environmental and consumer safety, and
generated numerous ethical
and legal discussions.
A GMO can be defined as alhewl'organisnl whch contains an
"intergenericcombinationofgeneticmaterial"
(HALLERMAN and
KAPUSCNSKI, 1990a) or as anorganismwhichcontains"genetic
material wlich has been modifiedi n vitro".
I will first explain the technological aspects ofgenerating transgenic
fish. The implicationsforaquaculture
are highlightedwith the
transgenespromoting prowth, loweringfi-eezingtenlperatureand
inducingdiseaseresistance. 1 willalsodiscussenvironmental
and
ln conclusion the future of GMOs is
consumerrelatedaspects.
presented.
The transfer of DNA seq~~ences
and genes in fish is being done for
three main pus-poses:
(1) to understand the molecular genetics sf development
(2) as a mode1 for mamalian, read human genetics
(3) to enhance aquaculture production
The first reason is rnotivated by fimdamentalresearch and is driven
amongst others by the desire to study the molecular biology of the
exly development sf zebrafish (ROSSANT
cand H O P ~ S1992)
,
and
medaka (ISFEAWA et al., 1997). Progress is such that a groeving
number of housekeeping and cell-specific genes are being isolated,
mapped, and the resdting phenotype characterised ( G W O et al.,
1996a).
The second reason is iduenced by the limitations of m a m & m
models to stuc& h m a n diseases. The reproductive biology of mice
sets serious limits to the exprimental s t u d y of early development,
incluehg the regulation of gene expression. ROSSANT
and HOPKINS
(1992) nicely review the eapabilies of zebrafish in this perspective.
In general, fish fertilise their eggs externally, have numerous
progeny, are easy to culture, and have short generation times. It is
possible to make isogenic lines in a sinde generation and to screen
for mutants in haploid progeny (POWERS,
1989). An example af a
promising application in medicine is the targeted study of
circulatory and cardiae defects in zebrafish.
Findly,tramgenesis offers prspctives for the aceeleration of
selection. Indeeel, the domestication of organisms with an ecsnomic
interest has been going on for tens of centuries. The process has
been achieved through carefulobservation,
drected breeding,
cdling and strain management.
But
fish
has a rather short
domestication history and thus the selectisn of strains and lines is
limited. Common carp has been selected in China since 2660 B.P.
( K o m , 1990), whle the selection of rainbow trout started only
last century and Atlantic salmon fourty year aga. Tropical fish such
as tilapia and caffish have been submitted to dlrected selection for
less than 10 year. A limiting factor is that selection progress is
L-
and
F. Volckaert
v35
F. Ollevier -fish
Transgenic
strongly dependent ongeneration time; for example bacteria double
every hour or so, while Atlantic s a h o n has a generation time of at
least 3 year. As such, transgenesis could speed up the generation of
lines with specific phenotypic traits: disease resistance, enhanced
growth, colour nwtation, and so on.
1Methodology
Regardless whether transgenic research is fundamental or applied,
three major steps have to be taken to generate transgenic fish. First
a recombinantexpressionvector has tobemade (l), second the
construct has to be introduced into the gernl line (2) and tlurd, the
desiredphenotype has tobe finctioning properly (3) (HACKER,
1993).
The construction of recombinant expression
vectors for fish
Expression vectors are recombinant DNAs that c a r y the transgene
of interest and the regulatory sequences thatdetemine where, when
andat which level the transgene will beexpressed(HACKETT,
1993). They include a vectorconsisting of a plasmid or viral
sequence, regulatory sequences andthe gene of interest.
Most vectors used are modified E. Coli plasnids ( c g . AMSTERDAM
et al., 1995; GIBBSet al., 1994; VOLCKAERT
et al., 1994), which
canbereplicatedin
E. eoli cellssuch that
amounts of
recombinant DNA can beobtained.
h
i
&
Retroviral vectors represent an alternative and
pronising strategy
(BURNS et al., 1993). Thegenotne of the Moloney Murine
Leukenlia Virus (MLV) was modfied with the envelope protein of
VesicularStotnatitisVirus(VSV).
The MLVNSV pseudotype
retroviral vectors showed enkanced sueeess sf integ-ation although
low viraltiters. A new generation o f
seriouslylinnitedbythe
MLVNSV pseudoptypedretroviralvectors has viral titers upto
1x1O'
colony forming units/mll \?rith an intepration success o f $3 %
( G M o et al., 1996b).
eeornbinant DNA
Regulatory elemenfs, promoters, enhancers
end dencers
About 3,000 genes pernit each ce11 toperfonn itsspcialised
Ruletion in a particular tissue. Consequently most of these genes are
non-hnctional nmst of the time and require precise signals to be
activated (HACKETT, 1993). Therefore genetic engineering requires
howledge of the elements wlich initiate, n~odifyand tenninate
protein
synthesis.
transc.ription, splice IIIRNA and initiate
Regulation of transcriptional initiation is the major control in gene
activation and is lnediated by a number of regulatory modules each
composed of a short DNA sequence and the proteins that
specifically interact cvith them. Tt is the combination of varisus
regulatorymodules.eachprovidingaresponse
(tissue-specific
expression, induction bya hormone, by a nletal ion, by a heat-shock
protein, etc) that cause the fine-tuning of gene expression. MI the
transcription factors bound to their DNA sequence and stabilised by
proteidprotein interaction,anchorto
the promoterof the RNA
polynlerase Il and are included in alargecomplexwithgeneral
transcription factors. Reviews have been Witten amongst others by
HACKETT (1993) and TYENGAR
et al. (1 996).
Numersus fish genes havebeenisolated,sequenced
and a lesser
number have beencharacterised,butthenumber
of regulatory
elementsisolated and sequenced is ratherlimited. Therefsre the
number of piscine sequences tested in vitro (in atransientlyor
stably transfected ce11 system) or in vivo (in a transient or stable
transgenic system) is linuted.
The ubiquitously regdated metallothionein (MT) promoter contains
Metal Responsive Elements (MRE) which2+ are DN& sequences that
mediateexpressioninthepresence of Zn and Cd (ZAFARULLAH
F. Volckaert and F. Ollevier - Transgenic fish
et al., 1989; FRIEDENREICHandSCHARTL,1990; HONG et al.,
1993). The interest of this promoter lies in its putative use as a
nlonitor of heavy metal pollution(KINOSHITA
et al., 1994).
The housekeepinggene p-actin has been characterised byL N et al.
(1990) and MOAVet al. ( 1993) in carp and TAKAGIet al. ( 1994) in
medaka. The latter authors developed an expression vector fiomthe
medakap-actin gene for use in medaka.Themedakap-actin
promotershowedvariable
but solnetinlesubiquitoustransient
expression in medaka(TAKAGI et al., 1994).Functionalanalysis
of the carp p-actin promoter in fish cellswas used to construct two
expressionvectorsfor use in transgenicfish (LIU et al., 1990).
Expression
is
species-specific
and
interactiolis
were
observed
betweenthemotifs in the proximalpromoterandfirstintron
in
zebrafish (MOAVet al., 1993).
(AFP) promotorlias
Thetype II andtypeIII-AntifieezeProtein
been fimctionally analysed il7 vitro and transiently in vivo (GONGet
al., 1991). Multiple positive and negative regulatory elenlents were
detected in this promoter which could be useful in controlling gene
expression of temperate species during cold temperature periods or
low temperature expression in tropical species.
The hormone prolactin 1 regulates various functions in fish such as
osmoregulation (fieshwater adaptation in saltwater fish and calcium
homeostasis
both
in
saltwater and fieshwater fish), Ca2+
metabolism,mucussecretion,hatclung,generalnletabolismand
behaviour. 117 vitro analysisof the regulatoryelenlentsofthe
prolactid gene of tilapia (Oreochronris n~ossa~~rbic~~s)
and rainbow
1nykis.s) includeseveralbindingsitesfora
trout (Ol?corly~~ch~rs
pituitary-specific
protein
(called
Pit-f), as well as positive
(enhancers)
and
negative
(repressors)
regulatory
elements
(ARGENTON
et al., 1996; PONCELETet al., 1996). A glucocorticoid
responsive element-like motifis also present close to the second and
tlurd Pit-1 bindingsites (ARGENTON
et al., 1996). Thetransient
expression of a fusion gene between the tilapia prolactin promoter
and a reporter gene in epithelial carp cells is dependent on the coexpression of the pituitary-specific Pit-l factor (PONCELETet al.,
1996). A transient in vivo study of deletion mutants of the tilapia
prolactin
promoter
revealed
the presence of enhancing
and
v 37
repressing
sequenees.
The location of tnaximl and ninimal
expression in vitro and in vnw eoincided to a lirnited degree.
Transgenes
Trmsgenes refer to the genas which are expresse$ in a transgenic
system, either za? vitro or in vivo, either transiently or stably. Their
propr expression is regulated by homologous orheterologous
promoter sequences. Three goups of transgenes can be dscerned.
(1) Transgenes
which
represent
a gain-of-function lead to
expression O a new protein in the eell. Examples are the gowth
hormone andthe anti-fieeze protein. (,2) The useofseleetable
markers such as the gene confienin3 resistmee to neomycin
facilitate the selection andidentification of transgenes. Weprter
genes such as those encoding j3-galactosidase, luciferase and green
fluorescent protein permit the examination of regulatory elements.
( 3 ) The expression ofloss-of-funetion transgenes interferes with the
normal funetioning of endogenous genes (HACKETT, 1993). A gene
encodng antisense niRNA that wouldprevent
ChRH protein
synthesis is under developnvmt.
Reviews on this topic have been published amongst others by
GONG
and HEW (19951, HACKETT (1993) and JYENGAR et a?. (1 996).
Gven the observation that in general ‘“all-fish-construets” p r f o m
the best in transgenic fish (BETANCOURT
et
19931, the currently
used gain-of-fimetion transgenes are vi1ptually al1 of piscine origin
md derive from the gene propar (@NA) or are cDNA sequences
with intron sequences and the polyadenylation signal added. There
are a number of well docunsented cases, of whieh transgenies for
gowth homona and antifieeze protein vil1 be diseussed later on.
The Xnrk oncoene eneodes a reeeptortyrosine lunase whch
causes melanomas and is loeated on the sex chro~~~osome
(SCHARTL,
1990).Injection
of the CWTkLXnlrk constructintomedaka
embryos indueed elnsbryonic tunlour fornsation in tissues nonnally
expressing the Xmnrk proto-oncogene ( W m E R et a?.?1994). The
system is used as a suecessfùl mode1 for oncogenesis.
Gonadotropin releasing hormone (GnRH) ha5 beencloned in a
number of fish including zebra fish, tilapia. rainbow trout and
eatfish. It is responsible for sexual maturation and as sueh draws a
and
F. Volckaert
F. Ollevier - Transgenic fish
lot of interest in its potential to induce sterility ESTRO ROM et al.,
1992).
Other transgenes of interest includelysozyme, an antibacterial
enzymefound in various tissues of vertebrates.Current interest
traces its origin to its isolation fiom rainbow trout by DAUTIGNY
et
al. (1991). The gene encoding the pituitaryexpressedhormone
prolactin has been cloned anongst others fiom tilapia, it has been
characterised in vitro by SWENNENet al. (1992) and stably
transferredto rainbow trout, in which its pllenotype is under
investigation (BRETON, pers. conm). The
insulin-like
growth
factor-1 (IGF-1) is regulated by GH and is implicated in the growth
promotingeffectsof
GH. It has beencloned,sequencedand
characterised anlongst others in Atlantic saIlnon (DUGUAY
et al.,
1992).
The second group of transgenes, the reporter genes, are meant to
facilitate the
study
of regulatory
elements
because
they
are
relativelyeasilyandreliablydetected.Theycanalsobeusedin
large screens of mutants
by
gene-trapping
(identification
of
unknowngeneproducts)andenhancer-trapping(identificationof
unknownpromoter sequences) (GAIANOet al., 1996a). They are
usually of non-fis11 origin and cany characteristics whichmakes
theln relatively easily detected.
lac2 originates
fiom
E. Coli and
encodes
the
enzyme
pgalactosidase
which
is detected in vivo, colorimetically,
histochemically,
illltllunohistochelllically
lutuinometrically
or
(MACGREGOR
et al., 1990). It is the nlost often used reporter gene,
is lughly dependable and can be used under a variety of conditions
(see ALAM et al., 1995; LIN et al., 1994; SEKECALI
et al., 1994).
False positives can be obtained because of staining of the eye lens
and endogenous P-galactosidase activity in the stomach.
Chloramphenicol acetyltransferase (CA7')is detected radioactively
with a lugll degree of sensitivity in transgenic tissue extracts and
illlllunolustochetllically (see STUARTel al.. 1990).However the
need to work radioactively and
the long protein half-life of 50 h
(VOLCKAERT
et al., 1994) represent major drawbacks.
Luciferase (lm) originates fiom Photinus p-vralis (firefly) and
encodes the enzyme luciferase which can be detected with photon
v39
acquisition systems (scintillation counter, luminometer, X-ray film
and ceD cawera) and innnunolustochet~lically(see GIBBS el al.,
1994; MAYERHOFER
et al., 1995; VOLCICAERT
et al.* 1994).It is
currently the lnost sensitive and one of the tnost simple systems to
use.Advantagesinclude the absence of endogenousbackground
activity- the simple polypeptide structure of luciferase, the linearity
e l al., 1994).
over a range of 1O" and the half-life of311 (SEKKALI
Green fluorescentprotein
(GFP) originatesfrom
the jellifish
Aeqrrorea victoria and produces a bri& green fluorescent product
(&VISTERDAM et al., 1995).It does notseemto be
underlight
hannful to the transgenic fish anddoesnotrequireexogenously
addedproduet.Moreover,spectrallyslliftedmutantshavebeen
et al., 1995).
isolated (DELAGRAVE
The tyrosinase enzyme which is involved in pignentation, has been
introduced in ~nutantsof lnedaka ~V~AT'SUMOTO
et al., 1992). The
involved
in
nlelanin
concentrating
honnone (MCM), also
pigmentation is another candidate reporter gene ( ALESTROM el al.,
1992).
Selectable markers such as neomycin phosphotransferase (neo), an
antibioticresistance gene, can be usedtoselectfullytransgenic
anilnals (see attenlptby ZELENINef al., 1991).However,mosaic
transgenic fish (the nlost co1m1on case i n parental populations) will
also be liilled despite ne0 expression in a limited number of cells.
This makes ne8 less suitable for transgenic research.
The thrd group of transgenes, called loss-of-iünction genes, can be
divided intwo: transgenes whose product interferes withthe normal
expression of another gene and transgenes that handicap the nonml
transcription of a gene through DNA insertion (enhancer-trap and
gene-trap). Although this approach has not been used that much in
fish for variousreasons. there are a fewcases.Transgenes that
produce antisense mRNA of @nRH to block the nom~alexpression
of GIIW belons to the first strategy ( ALESTROMet al., 1994).
BAYERand @AMPOS-QRTEGA ( 1992) developed a zebrafishline
withpreferentialexpression
in primarysensoryneurons,
which
showed al1 the characteristics of an enhancer-trap. More recent is
the insertional mutagenesis in chen~icallymutated zebrafish which
facilitates the cloning of genes corresponding to a specific
F.and
Volckaelt
F. Ollevier - Transgenic fish
phenotype (GAIANOet al., 1996a).Although the latter doesnot
fully correspond to thegene-trap qualification, it sets a standard for
tlus kind ofresearch.
Germline transmission
A second important phase in achieving stable transgenic fish is the
pernlanent establishment of the transgene in the host genome. The
transgene has to be incorporated in genncells to be heritable. It Ilas
to be mentioned that with current practices transiently transgenic
fish are the most likely outcon~eof any attempt to insert transgenic
constructs. In such case plasluid-likestructuresampli&
in the
nucleus but outside the chromosomesduring early embryonic
development and disappear sometin~es later during embryonic
development (GONG and HEw, 1995; IYENGAR
et al., 1996;
VOLCKAERTet al., 1994). Reviews on the techniquestoinduce
transgenes have been prepared anlongst other by
CLOUD (1990),
GONG and HEW (1995), HOUDEBINE
and CHoURRoUT (1991),
MACLEAN and RAHMAN (1994) and OZATO
et al. (1989).
The first developed, most efficient and still most colmnonly used
technique is micro-injection.
Essentially,
micropipette
a
is
introduced in the oocyte or the newly fertilised zygote and a small
amount of a physiological solution containing the DNA construct is
injected. In the first case the target is thenucleus (see OZATOet al.,
1989); in the second case the target is the cytoplasm because the
polar body can not be localised (WINKLERet al., 1991). Usually
about IO4 to 1O7 copiesoftheconstructareintroduced;lower
alnounts jeopardise transformation and higher amounts tend to be
toxic (107 construct copies is about equivalent to the DNA content
of the nucleus). Fish eggs are relatively large ranging fiom 1 mm
(zebra fish) to 7 111111 (Salmon),and show variousdegreesof
transparency due to the presence of yolk.Thecorticalreaction
influences the penetrationofthechoriontovariousdegrees.
Therefore
injection
occurs in the blastodisc or through the
micropyle shortly after fertilisation (VOLCKAERT
et al., 1994), or in
dechorionated eggs (CULP et d., 1991).DNAshouldideally
be
injected intothe nucleus or pronucleus ofthe egg so that integration
V 41
42 V
Geneiics and aquaculture in M i c a
in the host genowe is .Facilitated. If integration occurs during early
development(blastula), al1 cellshave a chance to be transgenic,
although tlis depends alss on the nature of the eonstnlct.
Spem incubationnlight seem to be the easiest wayto trmsfect
orgomisms, but the results are variable ( C H O U w 3 J T and IPERROT,
1992; KHOO et al., 1992). It is thought that DNA nnight adhere to
the ce11 wall and be introduced in the egg during fertilisation. An
alternative is the electroporation of the sperm (MÜLLERet al., 1993;
SYMQNDSel al., 1994).Short electrical pulsespenneatethe ce11
membrane
temporarily
and
thus
facilitate
the entry of
rnxxromolecules sueh as DNA into the cytoplasrn~. It allows totreat
numerous eggs simulataneously, is highly
reprsducible
and
elilninates skilled labour.
h
i
&
-
Gene bombardmentinvolvesthebombardtnent
of cellsby
velocity lllllcroprojectiles of sln~all tungsten particles coated with a
DNA constntct (ZELENINet al., 1991). Althou& highly succesfid
in plant tissues, the absence of a impermeable ceIl wall of cellulose
seem to inhibit transformation.
The inclusion of DNA inIipophylicparticles(lipofection)which
fuse with the ce11 membraneefficientlytransfectsbacteriaand
use withfish
sgenn and
eukaryotic cells. Yt is suitablefor
dechorionated embryos. Transient expression of lipofected e s s of
Afiican caffish has been docutnented by SZELEIet al. ( 1994).
Retroviralinfection has been reportedabove. Most remarkable is
that its efficiency i n transfecting is several orders higher than the
previsusly mentionedmethods. A drawback is that thetnodified
retrovirus has to be injected.
However i n al1 these methods, integration of the transgene in the
host gensme occurs at random. Homologous recotnbination, ~nostly
aining at selectivedisruption of a target gene orinsertion of a
reporter gene, is routinelyachievedin
lnice because of the
availability of totipotent embryonic stem cells(ES). In a first step, a
targeting vectorcontainingabout
8 kb of the gene of interest
interrupted by sequences encoding a selectable marker is introduced
into ES cells. Selection and genomic DNA analysis identify ES ce11
clones with the appropriate integration event:thesi are injected into
blastocysts to generate chimaeric animals.
F. Ollevier -fish
Transgenic
and
F. Volckaett
Method
Bombardment
Eledroporation
Lipofedion
v43
Ease of
Parental
protocol
expression
++
++
+
YES’
Y ES’
YES3
++
+++
+
Micro-injection
Retroviral infection
YES4
YESG
j
Stable
inheritance
YES‘
YESG
YES7
ITable I
The efficiency of various methods to deliver DNA as reported
in the literature for ease of protocol, parental expressionand stable
inheritance. ZELENIN et a/.. 1991; MÜLLERe l al., 1993; SZELEI et
al., 1994; Numerous reports, including VOLCKAERTet al.. 1994and
WINKLER et a/.. 1992; STUART et a/.. 1990 and OZATO et al.. 1986;
GAIANO e l a/.. 1996b: SYMONDS et a/.. 1994.
‘
’
’
If these have germ cells derivedfrom the recombinant ES cells, they
will transmit the genotype to tlleir progeny. No totipotent ES cells
are available yet in fis11 systems that wouldallowhomologous
recombination, but severallaboratories are investigatingexactly
that. To this purpose ce11 transplants anlong zebraflsh blastulae and
the production ofgerm-line chinlaeras (LIN et al., 19921, embryonic
stem ce11 culture (COLLODIet al., 1992; HONGand SCHARTL,
1996)
andsuitablevectors
for site-specificintegration ( I Z S V M et al.,
1995; BEARZOTTJ
et al., 1992) are under development.
Expression of the transgene
Correct expression of the transgene is of course the major aim of
transgenic integration. However, it is labour intensive to identify
stable transgenic fishwith reliable transgenic expression becauseof
the high level of clumaeras observed. Several strategies have been
in vivo detection of stable
developed
to
facilitate
selection:
integration on genonlic DNA extracted fi-om skin tissue of progeny
after proteinase K digestion ( KAWAKAMI and HOPKINS, 1996). 117
vivo detection of expression by assaying enzymatic activity of Luc
(MAYERHOFER et al., 1995), tyrosinase (I~~ASTUMOTO
et al., 1992)
and lacZ (Lm et al., 1994) and by assaying fluorescence of GFP
(AMSTEFDAM et al., 1995). Bther screening metho&such
as
fluorescence in situ hybridisationandhistochemical
staining to
verify expression are rather tiwe-consunling.
The fate of the DNA constntct is remarkable imunediately aller
corresponds moreto the
introduction(transientreplication)and
general expectations in the
long
t e m (heritable or stable
integ-ation).
The fate of the DNA upon introductionin the transgenic fish system
behaves differently fr01n mammals. Soon after being introduced in
the cell, the circular fbsion genes for111 head-to-tailconcatemers
while the linearisedfusion genes fom1 randomarrangements of
head-to-tail, tail-to-td and head-to-head concatemers in the nucleus
( I ~ G AetRal-, 1996; VOLCKAERT
et al., 1994). During the rapid
cleavage stages of early development the fùsion DNA is quickly
replicated at the timeofrapid DNA synthesis in the developing
embryoup to the 10'" or 1 1 '" ce11 division.However a-fter the
midblastulatransition,degradation
rates exceed replication rates
and DNA fragments of 200 bp long are formed. The pattern is
similx to other Iswer vertebrates such as .;Yenopus @RIOLEAU et
al., 1994).
Transient expression is in some cases tissue-specific (promoter of
the carp myosinheavychainisoform
in zebra fish andcaffish
(PYENGebR et al., 1996)) while nstin
others (forexample the
prolaetin promoterincaffish(personal
data) and theubiquitous
promoters (ALAM et al., 1996)). However, transientexpression
seems to be llighly iduenced by the embrysnal tissue. The yolk
syncitial layer shows the highest level of expression and thus may
studies using
quantative
intluence transient
expression
in
comparative analysis (.WELL~PVIS
et al., 1996).
F.
and
Volckaert
F. Ollevier - Transaenic fish
The long terrn fate of introduced DNA
Exogenous DNA tnay have integrated into the host's chromosolnal
DNA after survivingthe degradation process ofthe ce11 during early
development. The chances of parental integration Vary fiom absent,
over poor (most cases, e.g. MOUE et al (1992) and SHEARS et al.
(1991)) to 80 % (GAIANOet al., 1996a). The site of integration is
thought to be aspecific;oftenmultiple
sites of integration are
et al., 1996). Moreover transgene expression is
observed (IYENGAR
often impaired by the sequences flanking its integration site (the so
called position effect) since most genonic DNA is considered to be
transcriptionally inactive heterochromatin. Another in1portantfactor
silencing expression is de novo methylation of DNA (GIBBSet al.,
1994; IYENGAR
et al., 1996). Methylation of cytosine residues at
CpG sites in the protnoter
seem
to
be occurring.
Genetic
background of the host genome,species-specificdevelopmental
dynatlics andenvirorunentalinfluenceson
gene expressionmay
also playarole
(KAPUSCINSKI and HALLE-,
1991). Stable
of FI (a 50 % inheritance is
integration is provenbymeans
expected) and F2 (a 100 % expected inheritance is expected) test
crosses, Southern
blotting
of transgenic @NA,
selective
amplificationby the polylnerase chain reaction (PCR) of the
introduced sequence or fluorescence i n situ llybridisation of the
karyotypes.
1Transgenic fish in aqluacultlAre
Solnetinles the novelty of the genetic engineering of transgenic fish
has generated exaggetrated expectations in regards
to
its
applications among scientists, fish farnlers, administrators and the
general public.Delayedmaturation,enhanced
fieezing tolerance,
disease resistance and especiallyenhanced growthof transgenic fish
have caught the imagination. The following paragraphs will focus
on tlu-ee cases.In the next chapter the envirotunental, consunler and
etlzical aspects of transgenic fish are hipldighted.
v45
The growtln honnone (GH) gene has been cloned in at least 17 fish
speeies (WALLIS,1996). Tt encodes for the importantcireulating
peptide growth Inonnone, secreted in the pituitary and idhencing
draws
amongstotlwrsmetabolismandseawateradaptation.It
specialinterest because of its growth prol~mting action when
provided
exogenously
or transgenically. h t i a l attempts were
n~arredby psor inheritance (an exception is INOUE et al., 1990) and
expression, which has been rdated to the nature of the esnstruct,
the malsunaliau origin, metlnylationor the absence of regulatory
of chinook
intronsequenees.Presentlyan"all-fish"constnlct
sallmon GH undercontrol S.€ the ocean pout AFP pror~~oter
has
induced dramatic growth increases (DEVLINet cd., 1994b, 1995a,
1995b). On average the transgenic fisln were 1 O to 30 titnes heavier
than the control group at 15 nlonths of age. At present. it is not elear
to what extent the endocrinology (e.g. the titer of growth homone)
and growth rate correlate,nor how the excess GH bindsto the
receptors. Transgenie coho salnmn undement precocious parr-smolt
transformation during their first fall, about 6 tnonths in advance to
theirnon-transgenicsiblings.It
has to be mentioned that the
such as
unregulated overproductisn of GH inducessideeffects
cratuofacial abnonnalities (DEV.LIN
et al., 1994a), colsur change
(DEVLN et al., 1995b), excessive fat deposition, and SB on. Similar
been documented with growth
but less dramtic effectshave
lnon~lonedrivenby the metallothioneinpromoterin
coho sallnon
(DU el al., 1992) and pike ( GROSSet al., 1992).
Arctic fishes synthesize Antifreeze Protein (AFP) wluch Iowers the
fieezitng point of the plasma. AFP is synthesised in the liver and
then released in the circulatory system. The winter flounder type 1
AFP gene is seasonally regulated and contains a well characterised
promoter (GONG et al., 1991). Cotnmercial fish with the AFP gene
would be in a eonmercially advantageous position if they tolerated
watertemperaturesbelow
-0.7T. Attenlptsareunder
way to
transfer this gene into Atlantic salmon (SHEARS et al., 1991) and
F.and
Volckaett
F. Ollevier - Transgenic fish
gol&sh (WANGet al., 1995) such that their tolerance to fi-eezing
can be lowered.About 3% ofAtlanticsalmonembryosinjected
witll the AFP gene expressed AFP, although at low levels (10 to 50
p g / d insteadof 2-5 n g h l ) . Lnheritanceliasbeen
shown, but
iùrther research is required before a coinmercial systemis available.
To thecontrary F2 and FA progenyofgoldfish had a lowercold
tolerance.
Disease resistance
An importantaimoftransgenic
fis11 could be tocreate a barrier
against viral and bacterial infections. One only lias to think of
the
impactoffurunculosisinfectionsby
the bacteriuni Aeromonas
salmonzcida causing
high
fish
mortalities
lnfectious
or
arhabdovirusaffecting
HematopoeticNecrosis Virus (W),
Northwest Pacific salmonids. Several strategies are possible: either
a protein is produced transgenically for vaccination, either
genes are
incorporated that elilninate viral or bacterial infections. Lysozyme
lias been shown to be a potent inhibitor of gram negative bacteria.
Itsantibacterialactivity can be enllancedbyproteinengmeering
(
I
B
Wet al., 1994). A rainbow trout lysozyme cDNA has been
isolated ( DALITIGNY
et al., 1991 ) and spliced tothe ocean trout AFP
promoter. hltegration in the Atlantic sahnon genome is i n progress
(IYENGARet a/., 1996).
1 Biosafety
Concerns have surfaced in conjunction witll the lugh expectations
from fis11 with novel genes. Researchers,administrationsand the
public soon discovered that the new opportunities offered by fish
with new traitsincluded as wellpostive as negativeelements.
Comnlents
in
the scientific
literature
and
press
focus on
environmental issues, ethical issues and consumer concerns.
v 47
Central intheenGromenta1
concerns is the question whether
escaped transgenic fish will transfer their special genes to wild and
natural popdations and
whether
these
genetically
modified
organisms (GMOs) wi11 be selected for. Will GMOs establish
themselves pmanently outside the c011&~2denvironment of the
aquaculture facility? The question is the more crucial as a growing
number of wild species, including fish, go extinct due to habitat
destruction,
pollution,
introductions,
hunting
and
fishing. The
accidental or consciousrelease
of transgenic organisms may
influence thisprseess
and pose a realthreat
t0 the natural
ecosysteril.
Evidence for the introgession of transgenics are n0t available yet
and according t0 sonle authors the few transgenic fish released are
m0st unlikelyto
be selectivelyfavoured
for (KNIBB, 1997).
Tramgenie fish have been documentedto be released in nature.
Weil protected land-based hatcheries are operational for transgenic
Atlantic salmon in Canada and Scotland. Sotne of the enviromental
concems refer also to accidentalescapessince
transgenics are
usually generated from do~msticatedfish and thus may be held in
vslrious Ends of containment facilities (land-, ocean-or lake-based).
Contairunent may involve different options; biologieal containment
refers to strategies such as sterilisation and monosex populations,
chemicalcontainmentrefers
to the treattnent of effluents with
chlorination/deehlorination while physical containment varies -Prom
minilna1 options (a fish pen in theocean) to nlaadlml options (indoor
research laboratories with discontinususoutflow) (I(AP~scn\rs~.ir
and WLERRILAN,
1990). However it is and never will be perfeet;
the weakest link will detemine the chances t0 escape. Escapes of
Atlantic salmon off Nonvay have reachedlevels
wherethe
domesticated stock surpasses several times the 'L$rldstock. but legal
historie levels
of
escapes.
pressure has curtailed
these
Hybridisation between native and domestieated Atlanticsalmsn in a
monitored river system in Ireland has revealed that wild animals and
crosses between wild females withfanned fish have a h
i
& survivd
and fitness (CROSS,pers. corn.). Farnled fish are often less adapted
to the local situation, although their numeric donlinance tnay lmke
resources unavailable for the local stock. An interesting example of
reduced fihess is the higher risk to natural predation of
gowth
activity
hormone treated fish becauseof
the higherlevelsof
f "
and
F. Volckaett
F. Ollevier - Transaenic fish
(JONSSONet al., 1996). If introgression will happen, genes are
likely to pass through the domesticated males.In general, escapes of
transgenics draws similarity to the introduction of exotics,
especially if a non-nativespecies or strain is involved.
Another method to estimate the effect of transgenics is modelling
based on establishedspecies (GLIDDON and
GOUDET, 1995). Fitness
traits, gene flow, fixation of alleles and behaviour affect
the chances
that a transgenic escapee breeds with local stock. It is clear that
each transgenic linehas its OWI traits and thus will have to be dealt
with case by case. In addition, the stability of recombinant DNA in
the naturalenvironment is influencedbyselectionsince
in the
absence of selection the insert will disappear due to the costs of
carriage and expression of the recombinant (LENSKIand NGUYEN,
1988).
There may be other ways of reducing the risks of transgenic fish by
switching
on
the
transgenic genes demand”.
”on
Promoters / enhancers are such that transcription is only initiated
and terminated in selected conditions. An example is the antifieeze
promoterwhich is activated at low temperatures (GONG et al.,
1991).
If recommendatations are made towards genetically modfied
transgenics (GMO), the following aspects should be considered:
(1) 1s the phenotype s a c i e n t l y characterised? 1s another trait
inducedthananticipated?
Has the molecularbehaviourof
the
promoter/enhancerand the gene beenanticipated? How many
copies are integrated? Does the DNA construct induce novel gene
combinations? 1s inheritance stable? 1s the retrovirussufficiently
specific?
(2) Are there sufficient physical, chemical and biological barriers
present in the experimental facility such that escapes are virtually
impossible. Othemise said, is the risk sufficiently assessed? What
is the weakest link in the system (HALLERMAN and KAPUSCINSKI,
1995)?
(3) If suf3cient information has been collected on the GMO in step
(l), has it been part of a quantitative breeding program such that
thenovel trait can be fùrther characterised andselected for?
lnbreeding of GMO’s is of concem.
Y49
(4) Does the custolner run a r i s k when eating transgenic fish?
In general policies are under review amongst others in the USA,
Canada, the EuropeanUnionand
Norway (WLERMAN
and
KAPuscmsIcr, 1995).
Ethieal aspects and consumer acceptanee of @MO’Sinterhine and
pose a serious pressure on the fish industry. h general the publie is
not in favour although the response depends on the socio-culturd
background of the customers. A British sunrey of customers views
on the acceptability of transgenie fish in fish famling showed that
36.5% had no opinion, 34.8% thought it was not acceptable and
28.7% thought itwas acceptable (PENMANet al., 1995). The
Canadian industry has reacted with the utnsost eare such that only
one hatchery on the East Coast has taken the challange to breed
transgenic Atlantic salmon.Food safety will have to be addressed to
avoid that non-desirableside-effects are propagated (BEKOWITZ
and KRYSPIN-SORENSEN,1993; CHATAKONDI ef al., 1995).
Patenting of transgenie fish has been envisaged (HALLERMAN and
~(B~US~TNSK
1990b).
I,
If suceessful patents are developed, the fish
food industry will likdy be dominated by a few con~paniesable to
invest in the considerable R&D costs.
Transgenic fish have entered the international forum and will stay
there. Itis elear that their value as researchtoolto
study the
regulation of gene expression, including the nmlecular bioiogy of
developmentandmolecularendocrinslogy
is extremelyvaluable.
Several genes have been underscruteny thanks to this approach and
many more are awaiting. It is only the question how many of the
10,000 housekeeping genes and 2,000 or so regulated genes will be
shdied to a certain levelof detail.
Solne of thegenes isolated will be suitable for applications, eitheri n
a biomedical or an agricultural (ineluding aquaculture) eontext. GH
F.and
Volckaelt
F. Ollevier
- Transgenic fish
V 51
has proven sonle potential, but because of ilnperfect knowledge of
its regulation and action, much more research remains to be done.
Other applications such as fish as bioreactor (to produce selected
proteins inbu&} have potential, although not nluchhas been proven
at the moment. The current DNA constructs are surely not optimal.
Furtherfinetuning
of the promoter/enhancerregiontocontrol
expressionandtheflankingregionstoenhancespecificity
of
integration are urgently
needed.
The research comnunity is
targeting this topic(TYENGAR
et al., 1996).
Another aspect which requires attention is the introduction of the
DNA construct.Theaim
is specificity, but thelackoffish
embryonic stem cells for homologous recotnbination
has meant a
serious drawback. Recent progress with modified proviruses looks
promising (GAIANOet al., 1996b) but suffers fkom poor
a
be
understanding of fishvirology. Future researchwilllikely
redirected towards this field.
An issue which has been somewhat neglected is the introduction of
GMOs in current selection programmes. Producers will surelywant
to continue to tnake progress
with selection of traits such as growth,
food conversion, disease resistance and so on. How the transgene
will be introduced in the population and tnaintained renlains to be
proven.Hybridisation is oneoption, the production of purelines
another one. Also, the question of multi-locus interactions of DNA
inserts
and
envirorunent-transgene
interactions
have
not
been
addressed.
Environtnental and consumer issues will likely dominate the public
forum in thecoming years. GMO’s are newandSocietydoesn’t
necessarilyhaveaclearanswerto
such newissuessurfacing.
Legislation is under continuous review (reflecting the dynanlic state
of GMOs) by national and multinational organisations including
the
Organisation for Econonllc Cooperation and Developnlent(OECD),
the International Councilfor the Exploration of the Sea (ICES), the
Food and Agriculture Organisation (FAO) and the United Nations
educational, scientific and cultural organisation (Unesco}.
GMOs
Most likely econonllc factors and consumer acceptance of
will deteruine large scale developments. At the molnent progress is
nlostlydrivenbyascientificpush,
but interestby the econonlic
comnunity is growing steadily.
.
Acknswledgements
F.V. is a research associate atthe Fund for Scientifc Research (FWO
Vlaanderen). Research report4 in this review kas been funded
by the European Union (gant BIOT CT90-0188),FWO
Maanderen (gant G2014.94) and tkeKatholieke
Universiteit Leuven (gant OT/90/16).
A. Belayeav (K.U.Leuvm) kindly
made UsefiIl cemnernts.
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