Hobbs, Rodney Stephen (2005) The ocean pout (Macrozoarces americanus) antifreeze protein gene promoter drives expression of antifreeze protein and growth hormone genes in transgenic Atlantic salmon (Salmo salar). Masters thesis, Memorial University of Newfoundland.
- Accepted Version
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Expression of the type Ill antifreeze protein (AFP) gene from ocean pout (OP5α) was examined in ocean pout and Atlantic salmon transgenic fora truncated version this gene (t-OP5α). Also, Atlantic salmon transgenic for an “all-fish chimeric gene construct” composed of the ocean pout type Ill AFP gene 5' and 3' ends linked to the Chinook salmon growth hormone (GH) gene (EO-1α) was examined for GH mRNA. Northern blot analysis of ocean pout demonstrated that AFP mRNA was detected in most tissues with extremely high levels observed in the liver, and high levels in the stomach and gill. Low AFP mRNA levels were observed in skin, mouth skin, intestine, spleen, and kidney, while barely detectable levels were observed in heart, ovary, brain, and blood cells. No AFP mRNA was observed in muscle. For t-OP5α transgenic Atlantic salmon, AFP mRNA was detected in most tissues with high levels observed in heart, liver, stomach, and brain, low levels observed in mouth skin, intestine, spleen, gill, and muscle, and barely detectable levels observed in kidney, ovary and skin. No AFP mRNA was observed in blood cells. Northern blot analysis of EO-1α transgenic salmon demonstrated that expression was observed only in pituitary and spleen, with moderate expression detected in the pituitary and low expression in the spleen. Reverse transcription polymerase chain reaction (RT-PCR) demonstrated that the OP5α gene is expressed in all tissues of the ocean pout, and all tissues except for blood in both transgenic Atlantic salmon lines (t-OP5α and EO-1α). Blood plasma samples from the three strains of fish were tested for AFP activity by determining the thermal hysteresis (TH) for each. TH measurements from winter acclimated ocean pout were high (approximately 1.36°C) with typical bipyrimidal ice crystal shaping indicative of a high amount of AFP, while t-OP5α transgenic fish showed little(< 0.01°C) TH activity, but yielded hexagonal shaped ice crystals indicative of the presence of AFP. On the other hand, EO-1α transgenics showed no (0°C) TH activity and no ice crystal shaping, as expected. The differences observed in the level of expression between ocean pout and the two transgenic fish lines may be attributed to gene copy number, where it is high in ocean pout and low in transgenic salmon, and promoter integrity, where the promoter is intact in ocean pout and truncated in both transgenic lines. In addition, differences in expression levels between the two transgenic lines may be a result of the presence or absence of intervening sequences (introns) in the DNA where the t-OP5α construct contains introns while the EO-1α construct does not. Finally, it is apparent by its ubiquitous expression in transgenic Atlantic salmon that, unlike the type I AFP from winter flounder, the introns within the ocean pout OP5α AFP gene do not contain tissue specific elements, or salmon lack the factors needed to recognize them.
|Item Type:||Thesis (Masters)|
|Additional Information:||Bibliography: leaves 62-69.|
|Department(s):||Science, Faculty of > Biology|
|Library of Congress Subject Heading:||Antifreeze proteins--Newfoundland and Labrador; Atlantic salmon--Newfoundland and Labrador--Genetics; Ocean pout--Newfoundland and Labrador; Transgenic fish--Newfoundland and Labrador.|
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