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Aquaculture industry
The Atlantic coast of Canada is characterized by 0 to -1.8°C water temperature. Salmonid fish freeze when they come into contact with ice at temperatures below -0.07°C. This limits sea-cage aquaculture to the southern regions of the Atlantic. Initial studies were intended to understand the properties of antifreeze proteins to engineer freeze-resistant fish. The purpose was increasing the expansion of the aquaculture industry in the northern Atlantic. AquAdvantage salmon was developed to improve the economics of fish culture activities by increasing the growth rate and improving the feed conversion efficiency (Fletcher & Davies, 1991)

Antifreeze protein
Antifreeze genes are normally expressed in the liver, and the protein has to be secreted into the extracellular spaces to act in other tissues. Early studies also shown that one of the difficulties of transgenic expression of antifreeze proteins is that antifreeze genes contain up to 150 copies. This is further complicated because there is a strong correlation between gene copy number and the level of antifreeze protein naturally expressed in fish (Hew et al., 1988)

Growth hormone
Blood growth hormone levels in fish are naturally very low, less than 50 ng/ml. To enhance growth rate, it is not necessary for the gene to have a strong promoter. Nevertheless, it is necessary to modify the tissue-specific expression. One of the solutions to overcome this limitation is using promoters expressed in other tissues different from the naturally occurring pituitary gland (Du et al., 1992).

Timeline
The first genetically modified animal with enhanced growth rate was a mouse in 1974. It used a rat growth hormone gene driven by a mouse metallothionein promoter. Experiments to enhance growth rate in fish by manipulating growth hormone levels started around 1980's (Fletcher & Davies, 1991). To overcome the limitation of growth hormone expression in the pituitary gland, chimeric constructs using different promoters were tested. It included mouse metallothionein-1, Rous sarcoma virus (RSV) and SV40 (Du et al., 1992). Human growth hormone was also included in several constructs. Some studies found that mammalian constructs had little effect on fish (Fletcher & Davies, 1991). Concern was raised about potential health hazards and consumer acceptance by the effect of heavy metals of metallothionein promoters and also of viral promoters. The use of human and other mammalian growth hormone genes encountered regulatory obstacles. By 1989, a construct containing the promoter and terminator sequences from the ocean pout and the growth hormone of the Chinook salmon (opAFP-GHc2) was already developed and injected into Atlantic salmon eggs. By 1990 the genetic construct was detected by PCR in the nucleated red blood cells and gill tissue of selected individuals (Du et al., 1992) The results of the stability of the genetic construct up to the 4th generation was successfully confirmed and published by 2006 (Yaskowiak et al., 2006). Previously, the stability of other genetic constructs containing the metallothionein-B promoter fused with the full sequence of the type -1 growth hormone gene (pOnMTGHl) was confirmed in the in the sockeye salmon in 2004 (Devlin et al., 2004). This concept was similar to the one employed to stimulate growth in the transgenic mice. The genetic construct (pOnMTGHl) was injected into the blastodisc region of coho salmon eggs. It was found that 6.2% of the individuals that reach one year of age retained the transgenic DNA. When testing fin tissue and blood cells, this first generation were found to be chimeras. In addition to the enhanced growth rate, the transgenic coho salmon displayed precocious phenotype development for the spring migration from the fresh water to the marine environment. These animals also exhibited high unregulated levels -up to 40-fold- of the growth hormone during the winter (Devlin et al., 2004).