52 Part I: Principles
hormone (GH) gene from Chinook salmon (Zbi-
kowska 2003). The main obstacle to be overcome
for the achievement of this goal is to better under-
stand the potential risks involved with the release of
transgenic fish in the wild, and at this point, not
enough research has been conducted to answer these
concerns (Muir and Howard 1999). One option to
avoid proliferation of transgenic fish in the wild is to
sterilize all transgenic fish, but a reliable method for
100% sterilization has not yet been achieved (Razak
et al. 1999). Some of the transgenic strategies that
are being developed to improve growth rate and
increase the antifreeze property are described below.
IMPROVINGFISHGROWTHRAT E
The fish growth hormone gene has been cloned and
characterized from a number of fishes, including
many salmon species (Du et al. 1992, Devlin et al.
1994). Researchers from the University of South-
ampton in the United Kingdom (Rahman et al.
1998) developed transgenic tilapia fish (Oreo-
chromis niloticus) transformed with growth hor-
mone genes of several salmonids. Rahman et al.
used different types of constructs in their experi-
ment, but the one that gave the best results was the
construct with a Chinook salmon GH gene under the
control of the ocean pout antifreeze promoter. The
method used for the insertion of the transgene con-
struct was the cytoplasmic microinjection of fertil-
ized fish egg. The researchers reported the success-
ful genomic integration of the construct in the
founder (G0) tilapia and subsequent transfer of the
transgene to the G1 and G2 generations. Transgenic
tilapia expressing the transgene showed a growth
rate three times greater than the wild-type tilapia
and had a 33% higher food conversion ratio, which
would reduce farmers’ production cost. This trans-
genic tilapia also showed infertility at mature age,
which is a desirable trait for commercial transgenic
fish (Rahman et al. 1998).
INCREASINGANTIFREEZEPROPERTY INFISH
Many species of fish, such as ocean pout (Macro-
zoarces americanus) and winter flounder (Pleu-
ronectes americanus),that inhabit below-freezing
water in the northern regions produce and secrete
specific proteins in their plasma to protect their bod-
ies from freezing (Davies and Hew 1990). To this
date, two types of these proteins that have been char-
acterized are the antifreeze proteins (AFPs) and
antifreeze glycoproteins (AFGs) (Davies and Sykes
1997). AFPs and AFGs lower the freezing tempera-
ture of the fish’s serum and therefore protect the fish
from freezing by attaching themselves to the ice
surface, inhibiting ice crystal formation (DeVries
1984). There are four types of AFPs (I, II, III, IV)
and at least one type of AFG identified at this time
(Davies and Hew 1990, Deng et al. 1997). Most
aquaculture-important species of fish, such as the
Atlantic salmon and the tilapia, do not naturally pro-
duce any type of antifreeze protein and therefore
cannot survive and be raised in areas of the world
where water reaches sub-zero temperatures, which
creates a major problem for sea cage farming along
the northern Atlantic coast (Hew et al. 1995). The
production of commercially important transgenic
fish, especially salmon, that are freeze tolerant would
greatly expand the area for fish farms, increase pro-
ductivity, and reduce prices for consumers.
Flounder AFPs are small polypeptides that are
part of the Type I AFPs, which have two different
isoforms, “skin-type” and “liver-type.” Skin-type
AFPs are intracellular, mature proteins expressed in
several peripheral tissues; liver-type AFPs are im-
mature proteins that need to be further processed
before being secreted into circulation and are found
mainly in the liver tissue (Hew et al. 1986, Gong et
al. 1996). Hew et al. (1999) used the liver-type AFP
gene from winter flounder to generate a transgenic
stable line of Atlantic salmon (Salmo salar)that
demonstrated freeze tolerance capacity. By injecting
the genes into the fertilized eggs, a single copy of
the AFP gene was inserted and integrated into the
salmon chromosome, generating transgenic founder
fish with stable AFP expression and biologically
active protein. The same levels of expression and
protein activity were observed in up to three subse-
quent generations of transgenic salmon. Expression
of AFP was liver specific and demonstrated seasonal
variations similar to those in winter flounder, but the
levels of AFP in the blood of these fish were low
(250 g/mL) compared with natural AFP concentra-
tions in winter flounder (10–20 mg/mL) and there-
fore insufficient to provide freeze resistance to the
salmon (Hew et al. 1999). The focus of current
research has been to design gene constructs that will
increase the copy numbers of the transgene and
therefore enhance expression levels of AFP in ap-