Presentation on theme: "The Growth of growth hormone transgenic coho salmon Oncorhynchus kisutch is influenced by construct promoter type and family line."— Presentation transcript:
The Growth of growth hormone transgenic coho salmon Oncorhynchus kisutch is influenced by construct promoter type and family line
Background Coho salmon Growth hormone (GH) GH Transgenic Research Significance Methods Results conclusion and further research
Kingdom: Animalia Phylum: chordata Family: salmonidea Genus: oncorhychus Species : O. kisutch Behavior: migrate from a marine environment into freshwater streams and rivers of their birth; they spawn only once and then die
stimulates tissue growth R.A. Leggatt et al. (2012) Aquaculture 356–357 dairy production in the USA and the potential uses of GH are currently being explored in aquaculture, including the alteration of GH gene expression in fish by Transgenesis. Such research has created salmon that can average up to 11-fold heavier than normal fish after 1 year of growth
the process of introducing an exogenous gene – called a “Transgene” – into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. http://en.wikipedia.org/wiki/Herman_the_Bull
The worlds population is expected to rise from 6.9 to 9.3 billion until 2050. Fish are the main source of protein for billions of people around the world Rising standard of living = massive consumerism of meat products and sea food Decline in fish catch in the past decade. High commercial value
this technology has been explored in fish to develop enhanced strains for aquaculture, and for use as models to examine physiological controls of growth and energy utilization. R.A. Leggatt et al. (2012) Aquaculture 356–357
rapid growth has been found in some cases to cause abnormalities in transgenic salmon indicating that generating strains with the appropriate level of GH expression may be critical to avoid negative effects while also accelerating growth. Leggatt et al. (2012) Aquaculture 356–357
Studies have shown that growth acceleration is most pronounced if promoter and structural gene are homologous to the target organism's genome.
Metallothionein -B (MT), histone-3 (H3), promoters from sockeye salmon (Oncorhynchus nerka) were previously isolated and tested in vitro. Their constructs with GH1 showed strongly accelerate growth in Coho salmon (O. kisutch), Arctic charr (Salvelinus alpinus), and rainbow trout (Oncorhynchus mykiss) Leggatt et al. (2012) Aquaculture 356–357
The MT promoter can be stimulated by some heavy metals The use of MT as a promoter in areas where water contains high metal content may result in unforeseen effects artificial salmon diets are supplemented with zinc which likely influences expression relative to natural non-formulated diets. the effectiveness of the sockeye salmon H3 promoter coupled to the GH1 gene in increasing growth of Coho salmon warrants examination as an alternative to the sockeye MT promoter. Leggatt et al. (2012) Aquaculture 356–357
Creation of transgenic strains microinjection methodology Constructs synthesis & PCR Acromegaly test southern blot analysis pressure shocking to create triploid fish Plasma hormone levels GH and IGF-I levels analysis Data analysis Statistic methods
Transgenic Coho salmon were produced from the Chehalis River wild females using microinjection methodology. the process of using a glass Micropipette to insert substances at a microscopic level into a single living cell. This process is frequently used as a vector in genetic engineering and transgenesis to insert genetic material into a single cell
Two transgenic constructs were used, OnMTGH1 consisting of a MTB (Metallothionein -B) and OnH3GH1 (H3,) Leggatt et al. (2012) Aquaculture 356–357
Devlin et al (2004) Aquaculture 236 607–632 A B
observed in G0 fish prior to maturation. Acromegaly of these fish was assessed and rated on a scale of normal morphology, or mild, medium, or severe acromegaly. F1 transgenic fish were created from wild type strains and transgenic fish that reached sexual maturity.
to determine approximate quantification of the GH transgene 2-5 individuals of each strain were tested by southern blot analysis. Sockeye salmon GH2 was used as a probe (binds to all GH genes). strains were grouped into 3 categories based on intensity of the sockeye GH1 band.
The MT and H3 strains were propagated by mating F1 transgenic fish with multiple (greater than ten) wild-type Chehalis River fish. F3 generation siblings were produced by crossing F2 hemizygous transgenic fish with wild-type Chehalis River Coho salmon gametes with a single transgenic individual. Groups of fish were marked by promoter type, partial passive integrated transponder (PIT) tag numbers of the original G0 transgenic parent, and whether transgenic parent was male (M) or female (F) where appropriate.
Triploidy was induced in a subsample of two males one with MT promoter and one with H3, using pressure shocking of newly fertilized eggs. The Centre for Aquatic Biotechnology Regulatory Research (CABRR)
Transgenic fish and non-transgenic siblings (age- control) were reared in fresh well water (10±1 °C) Transfer to seawater when signs of smoltification start to appear. (temperature 8–14 °C). Genotypes of animals were tested using a transgene -specific PCR test fish kept in the small (200 liters) tanks during their freshwater growth phase. Medium (3000 to 5000 liters) tanks in which fish are kept during their seawater phase. Genotyping analyses in the molecular biology laboratory include assays for presence of transgenes.
intraperitoneal Passive Integrated Transponder (PIT) tags implanted at initiation of growth trials. .Weight and length were monitored from day 290 to 743, at which time the fish sexually matured and died. Navarro(2006) Aquaculture, volume 257, 309–315
Somatic growth is mediated through the GH/insulin-like growth factor-I (IGF-I) axis hormonal pathway Côté et al (2007) BMC Genetics
Samples were taken in the fall at day 290 (n=10 for each strain). Plasma GH and IGF-I levels were analyzed in transgenic salmon strains as well as size-matched non-transgenic control fish. Blood was collected in capillary tubes and centrifuged for 5 min at 4 °C. Plasma was the collected, frozen, and stored at −80 °C for radioimmunoassay measurements of GH and IGF-I. Leggatt et al. (2012) Aquaculture 193–199
Condition factors (CF) were calculated as: CF=100×(weight)/(length) 3 When values are high=good environment conditions. Data was analyzed using statistic methods
Range of GH transgene copy number based on southern blot band intensity in F1 fish transgenic for the OnMTGH1 or OnH3GH1 transgene Leggatt et al. (2012) Aquaculture 193–199
significant differences in abilities to promote growth in defined stable lines of GH transgenic Coho salmon were found. Both the OnMTGH1 and OnH3GH1 gene constructs dramatically increased growth of transgenic Coho salmon relative to non transgenic fish.
H3 strains had overall lower weight gain than MT strains. This indicates that H3 is a less strong promoter than MT for the sockeye salmon GH1 gene in vivo in Coho salmon. Fast growing strains sowed more abnormalities affects. accelerated growth in transgenic fish appears to be due in part to an up regulation of GH action through increased circulating IGF-I levels.
Copy number of GH transgene did not appear to influence the differences in growth rates between strains Differences in growth rate between strains within each construct could be due to location of GH insertion in the founder parent's genome, which could influence the degree of transgene expression