2012 Annual Report
1a.Objectives (from AD-416):
1: Develop homozygous clonal lines of rainbow trout resistant to bacterial and viral pathogens from previously developed transgenic trout. Test disease resistance in outcrosses of different genetic background to evaluate protection.
•1.a. Develop homozygous clonal lines of disease resistant rainbow trout.
•1.b. Identify the expression profiles of genes related to innate immunity and adaptive immunity pathways in homozygous clonal lines of transgenic fish resistant to bacterial, parasitic, and viral infections.
2: Assess whether transgenic rainbow trout resistant to bacterial and viral pathogens are also resistant to infection by parasites (e.g., Ceratomyxa shasta and Myxbolus cerebralis).
3: Develop transgenic rainbow trout capable of accumulating astaxanthin in the flesh and identifying and characterizing factors affecting this trait.
1b.Approach (from AD-416):
With the rapid growth of the human population and the increasing consumption of seafood for health considerations, the world demand for seafood products is mounting rapidly. To this end many countries have turned their attention to various forms of intensive aquaculture production. However, intensive aquaculture has brought about several negative impacts such as increasing disease outbreak incidences and decreasing the quality and nutritional value of fish flesh, resulting in significant economic losses. The application of modern molecular biological technologies including genomics and proteomics will enhance efforts to resolve these problems. We have developed transgenic trout carrying disease resistance genes known as cecropins: these fish exhibit resistance to infection by pathogens affecting aquaculture production including Aeromonas salmonicida and infectious hepatic necrosis virus (IHNV). Using disease resistant transgenic fish and new strains capable of accumulating higher levels of astaxanthin in the flesh as experimental models, we propose to identify and characterize specific pathways and genes leading to increased disease resistance and improved flesh color and nutritional quality. Genes identified from these studies will be characterized for their potential in developing superior broodstock through selective breeding.
Objective 1. DNA microarray analysis was used to identify the gene expression profiles in the innate and adapted immunity pathwaysof rainbow trout. Multiple high quality RNA samples were isolated from kidney, spleen and liver tissues of two transgenic families of fish and non-transgenic fish. The results showed that a total of 1000 genes showed up-regulation in spleen tissue of transgenic fish when compared with spleen RNA of non-transgenic fish. Similar results were observed in liver RNA and kidney RNA samples. Additional analyses were conducted to confirm the expression levels of genes associated with innate and adaptive immune pathways.
Objective 2. New transgene constructs containing crtW and crtZ genes were constructed and were introduced into rainbow trout sperm by electroporation, and the resulting sperm was used to fertilize 2000 rainbow trout eggs. About 200 fertilized eggs hatched and the resulting fry were raised to adulthood. Fin tissues were collected for genotyping.