1a.Objectives (from AD-416):
1) Develop homozygous clonal lines of rainbow trout resistant to bacterial and viral pathogens from previously developed transgenic trout and test disease resistance in outcrosses of different genetic background to evaluate protection.
2) Assess whether transgenic rainbow trout resistant to bacterial and viral pathogens are also resistant to infection by parasites (e.g., Ceratomyxa shasta and Chthyophthirius multifiliis).
3) Develop transgenic rainbow trout capable of accumulating astaxanthin in the flesh and identifying and charactering factors (genes) affecting this trait.
1b.Approach (from AD-416):
Using the disease resistant transgenic trout and the transgenic trout 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.
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 nontransgenic 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.
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.