2011 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 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.
We have bred the following clonal lines of cecropin P1 transgenic fish: S7-342-F695, S8-505-G231, S7-375-F180, S9-746-F509, S9-659-F073, S9-638-F297, U6-768-G410, A12-944, and A13-831). The disease resistance characteristic of each clonal line has also been confirmed by challenge studies against Aeromonas salmonicida and IHNV. These are all male fish and the sperm samples collected from multiple individuals of each line were cryo-preseved. Tissues of kidney, liver, and spleen from several clonal lines have been collected and RNA extracted. DNA microarray analysis is under way to determine the profile of gene expression of innate immunity and adapted immunity related genes.
Although crtW and crtZ genes obtained from our collaborator, Dr. Y.T. Kim of South Korea, were cloned into transgene vector and transformed into CHSE cells, synthesis of astaxanthin was not detected. After many nucleotide sequence determination attempts, it was concluded that both crtW and crtZ genes were incorrect. For this reason, we were not able to carry out gene transfer into rainbow trout. Now we have re-isolated crtW and crtZ genes from Paracocus haeundaensis. A di-cistronic transgene containing crtW and crtZ genes has been constructed. This di-cistronic transgene will be introduced into rainbow trout in the new fiscal year.
Heterozygous fish from several clonal lines carrying pig cecropin P1 gene have been established. Challenge studies with parasite Ceratomyxa shasta will be conducted in the spring 2012.
To date nine clonal lines of disease resistance transgenic trout have been produced. The sperm samples of these fish have been cryo-preserved.
The ADODR is in frequent contact with the cooperator through phone calls, email, and site visits in addition to receipt of written reports.