By definition, transgenic or genetically modified organisms (GMOs) are those that have had foreign DNA (deoxyribonucleic acid) artificially inserted into their own genomes (Chen and others 1996; FAO 2000). The first successful case of transgenic fish was reported in 1985, when Zhu and colleagues microinjected the human GH gene into the fertilized eggs of goldfish (Carassius auratus L. 1758) (Zhu and others 1985). This was followed by successful introduction of human GH gene into the genome of the loach (Misgurnus anguillicaudatus) with resulting transgenic fish that grew 3 to 4.6 times faster than the control within the first 135 d (Zhu and others 1986). Since 1985, the field of transgenics has experienced a number of technological advances. Many genetically modified fish species have been established along with various methods for foreign gene insertion (such as microinjection, electroporation, infection with pantropic defective retroviral vectors, particle gun bombardment, and sperm- and testis-mediated gene transfer methods) and detection (such as polymerase chain reaction [PCR]-based assay and Southern blot analysis) (Chen and others 1996; Lu and others 2002; Sarmasik 2003; Collares and others 2005; Pandian and Venugopal 2005; Smith and Spadafora 2005). Transgenic fish are appealing to some because attainment of desired traits is generally more effective, direct, and selective than traditional breeding, and could prove to be an economic benefit for improvement of production efficiency in aquaculture worldwide (Nam and others 2002; Ramirez and Morrissey 2003). In addition, fish species tend to be relatively tolerant to artificial manipulation of their genes during early development, making them ideal subjects for genetic modification (Foresti 2000). However, there are numerous environmental and human health concerns, which will be addressed later in this section, that are associated with the use of transgenic technology in aquaculture (FAO 2000; Fleming and others 2000; Aerni 2004; Naylor and others 2005).

To create a transgenic fish, a DNA construct containing genes for the desired trait(s) along with a promoter sequence is generally introduced into the pronuclear of fertilized eggs. This is followed by in vitro or in vivo (implanted into the uterus of a pseudopregnant female) incubation of the injected embryos and subsequent maturation into a fully developed transgenic organism (Chen and others 1996). Once transgenes have become integrated into a host organism’s DNA, they can be passed on to future generations (Chen and others 1996), with the possibility of 100% transmission using stable isogonics transgenic lines (Nam and others 2002). Zebrafish are often used as a model species in transgenic studies owing to a number of biological and experimental advantages: they are easy to maintain and breed, their embryos are sturdy against experimental manipulations, they develop rapidly, they have a large number of offspring, and they are transparent at some stages of development, facilitating the use of fluorescent transgenes (Dahm and Geisler 2006). Thus far, fish including Atlantic, Coho, and Chinook salmon, rainbow and cutthroat trout, tilapia, striped bass, mud loach, channel catfish, common carp, Indian major carp, goldfish, Japanese medaka, northern pike, red and silver sea bream, walleye, and zebrafish have been genetically modified to produce select traits (see Table 1) such as increased growth, increased feed conversion efficiency, cold tolerance, and disease resistance, all of which will be thoroughly discussed in the following subsections.

Growth hormone.
GH is a polypeptide that is excreted from the pituitary gland, binds specific cell receptors, and induces synthesis and secretion of insulin-like growth factors (IGF-1 and IGF-II), resulting in promotion of somatic growth through improved appetite, feeding efficiency, and growth rate (Hsih and others 1997; de la Fuente and others 1998). In fish, the central nervous system (CNS) normally controls GH excretion levels, which are highly variable, occurring seasonally and in bursts. However, the AFP gene of ocean pout (Macrozoarces americanus) is expressed year-round in the liver. As a way of bypassing CNS control on GH expression, transgenic research typically involves linking the GH gene to the AFP gene promoter (Fletcher and others 2004). Enhanced secretion of GH and subsequent fish size augmentation could greatly reduce production costs associated with aquaculture by reducing the time to market size and lowering exposure to risks such as disease and predators (Cook and others 2000). Although attempts to introduce GH exogenously have also been met with some success, commercial application of strains of fish containing transgenically introduced GH, once developed, could prove to be more cost-effective (Chen and others 1996). Increased growth has been the most thoroughly researched of the possible transgenically induced fish traits, and it is predicted that this technology will soon be applied to commercial aquaculture production (Wu and others 2003). However, a major remaining challenge is to determine the optimum balance of GH required for obtaining maximum growth without causing harmful effects to the organism (de la Fuente and others 1998), as excess GH can cause problems such as acromegaly (associated in humans with excess bony growth in the jaw) and head enlargement (Rahman and others 2001).

Since the initial transgenic introduction of human GH gene into goldfish and mud loach (Zhu and others 1985, 1986), extensive research has been performed on the use of GH in a wide variety of aquatic species. New, improved techniques have been developed for introduction of transgenes into host genomes, and there has been a focus on the use of GH genes originating from fish rather than humans, with the hope of increasing consumer acceptability (Levy and others 2000). Negative perceptions associated with the use of viral promoters to express transgenes have also driven many researchers to replace them with fish-based promoters for the creation of “all-fish” transgene constructs (Galli 2002). These all-fish GH-transgenic strains have been developed in a number of species, including common carp (Cyprinus carpio), silver sea bream (Sparus sarba), red sea bream (Pagrosomus major), tilapia (Oreochromis niloticus), and Atlantic salmon (Salmo salar) (Table 1).

As discussed by Wu and others (2003), an all-fish GH construct has been successfully introduced into the common carp, resulting in increased growth rate and more efficient feed conversion as compared to farmed fish controls. Middle-scale trials of these all-fish GH-transgenic common carp have shown high potential for successful commercial application in aquaculture. In 2003, carps and other cyprinids were the top species group produced worldwide in aquaculture, contributing 17.2 million tons (live weight) to the global food supply (Johnson 2005). Use of transgenic technology in carp aquaculture could prove to be highly beneficial to the industry and further increase the availability of these fish.

Transgenic lines of silver sea bream, an economically important cultivated species in Asia, were developed using a construct containing rainbow trout (Oncorhynchus mykiss) GH complementary DNA (cDNA) with a common carp promoter (Lu and others 2002). Using 2 novel methods of introduction, sperm- and testis-mediated gene transfer, between 56% and 76% of the animals carried the transgene, and several showed faster growth than controls (Lu and others 2002).
Red sea bream has high economic value in China, but production is limited by a relatively slow growth rate (Zhang and others 1998). With hopes of developing this industry, Zhang and others (1998) introduced an all-fish gene construct containing Chinook salmon (Oncorhynchus tshawytscha) GH with an ocean pout AFP gene promoter into fertilized eggs using electroporation, a technique that allows for treatment of over 10000 eggs in 10 min. After 30 d, 29% of the fish carried the foreign DNA sequence and after 2 mo, 38% were found to carry the sequence. Compared to controls at 7 mo, the average body weight and length of the transgenic individuals increased by 21% and 9.3%, respectively.

Tilapia is an economically important aquaculture species that is farmed in over 60 countries worldwide (Rahman and Maclean 1998). All-fish transgenic tilapia were successfully created by microinjection of Chinook salmon GH linked to an ocean pout AFP promoter. The transgenic tilapias were found to grow almost 4 times faster than nontransgenic siblings, and the transgenic sequence was successfully transmitted through the germline (Rahman and Maclean 1998). A more recent, long-term trial found a 2.5-fold increase in growth and 20% greater food conversion efficiency, with more efficient utilization of protein, dry matter, and energy, as compared to nontransgenic siblings (Rahman and others 2001). In an investigation into the expression sites of the AFP type III promoter/Chinook salmon GH construct in tilapia, messenger ribonucleic acid (RNA) containing the transgenic GH was expected to be mainly found in the liver (Caelers and others 2005). However, the resulting expression pattern was more reflective of natural rather than transgenic GH, with transgenic messenger RNA being detected in a number of other organs, including the gills, heart, brain, kidney, spleen, intestine, skeletal muscles, and testes. Since no growth abnormalities were observed, the authors suggested that this gene transfer method mimics the natural expression of GH in organs during development, resulting in growth-enhanced fish with normal proportions.

Transgenic tilapia containing the GH gene driven by the human cytomegalovirus (CMV) were compared to nontransgenic siblings on a number of metabolic and physiological parameters (Martinez and others 2000). The results showed several significant differences, with transgenic tilapia consuming 3.6-fold less food and having 290% greater feed conversion efficiency. In addition, growth efficiency, average protein synthesis, anabolic stimulation, and synthesis retention were higher for the transgenic tilapia. The authors also reported distinct metabolic differences for transgenic juveniles, concluding that GH-enhanced transgenic juvenile tilapia are biologically more efficient than nontransgenic juveniles.

Studies into the use of CMV as a promoter of the GH gene have also taken place with fish such as Arctic charr (Salvelinus alpinus L.) and the Indian major carp Labeo rohita (Pitkanen and others 1999b; Pandian and Venugopal 2005). Pitkanen and others (1999b) reported that CMV showed a greater ability to promote transgenic growth as compared with 2 piscine promoters, metallothionein B and histone 3. A further investigation into the use of CMV as a GH gene promoter showed no difference in the muscle composition of transgenic fish as compared to nontransgenic siblings (Krasnov and others 1999). However, the transgenic fish did show some metabolic features that are often observed in farmed salmonids, such as an increased metabolic rate and faster utilization of dietary lipids, particularly in the case of triglycerides. In transgenic studies involving the Indian major carp L. rohita, an element known as internal ribosomal entry sites (IRES) was included in an expression vector containing the CMV promoter and Indian major carp GH (Pandian and Venugopal 2005). This IRES element enables expression of 2 genes—the gene coding for the protein of interest (GH) along with a reporter gene (enhanced green fluorescent protein [EGFP]). Although the resulting transgenic carp exhibited 4- to 5-fold increases in growth compared to nontransgenic controls, 99% mortality was observed within 10 wk of hatching.

Transgenic Atlantic salmon containing Chinook salmon GH have also been developed (Du and others 1992; Fletcher and others 1992; Cook and others 2000). In 1 study, all-fish growth-enhanced transgenic Atlantic salmon produced by microinjection were found to have a 13-fold increase in body weight compared to nontransgenic controls (Du and others 1992). In a later study, all-fish growth-enhanced transgenic Atlantic salmon were found to grow at rates 2.62 to 2.85 times greater than nontransgenic controls, with a 10% greater feed conversion efficiency (Cook and others 2000). Transgenic salmon also ingested more, exhibiting 2.14- to 2.62-fold greater daily feed consumption, with lower body protein, dry matter, ash, lipid, and energy and higher moisture than nontransgenic controls (Cook and others 2000). The results of a recent study into the genetic expression and interactions of GH, IGF-I, and their receptors indicate involvement of the hormones in the areas of vertebral growth and bone density (Wargelius and others 2005). It was reported that plasma GH most likely activates growth in bony tissues, while IGF-I appears to be important in bone matrix production.

Fletcher and others (2004), whose laboratory began, studying GH gene transfer in 1989, reported successful expression and inheritance of the GH gene through 6 generations of Atlantic salmon. The transgenic salmon reach market size approximately 1 y earlier than nontransgenic farmed salmon. This technology was patented and licensed to Aqua Bounty Technologies™ of Waltham, Mass., U.S.A., which has recently announced the development of transgenic broodstocks of salmon, trout, and tilapia under the trade name of AquAdvantage™ (http://www.aquabounty.com/products.html). These fish, which contain the GH gene from Chinook salmon, express GH protein year-round instead of seasonally and are reported by the company to reach market size at least twice as fast as their nontransgenic counterparts (Dove 2005). The transgenic fish are also neutered to prevent interbreeding with wild stocks. AquAdvantage salmon, which grow at a rate 4 to 6 times greater than nontransgenic Atlantic salmon, are currently under regulatory review in both the United States and Canada (Castle and others 2005).
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