2010 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.
Using the technology of androgenesis, the following homozygous clonal lines of transgenic fish containing cecropin P1 constructs were created: S7-342-F695, S8-505-G231, S7-375-F180, S9746-F509 and S9-659-F-073 which are resistant to IHNV; and A. salmonicida, and S9-638-F297, U6-768-G410, A12-944 and A13-831 which are resistant to IHNV alone. Through genotyping and sex identification by PCR analysis, all male individual homozygous fish have been tagged. Individuals from families S7#375-F-073 and S9#659-F-180 have reached reproductive maturation. The other families will reach reproductive maturation by December 2010. The expression of cecropin P1 transgene in heart, spleen, muscle and liver of individual fish from S7#375-F-073 and S9#659-F-180 have been confirmed by RT-PCR analysis.
Sperm samples from S7#375-F-073 and S9#659-F-180 homozygous transgenic fish families have been cryopreserved.
Sperm of S7#375-F-073 and S9#659-F-180 were out-crossed to eggs from non-transgenic fish and the resulting offspring were challenged with Aeromonas salmonicioda. Results of the challenge studies showed that progeny derived from both families of fish exhibited resistant characteristics to Aeromonas salmonicidas.
A construct which contains crtW and crtZ genes driven by the Beta-actin gene promoter has been constructed. This double transgene construct is transfected into CHSE cells (Chinook salmon embryonic cells) to test for conversion of Beta-carotene into astaxanthin by HPLC analysis. The transgene has been proven to be functional. The transgene insert will be introduced into rainbow trout via electroporating the sperm following conditions developed in our laboratory. The resulting fish will be raised to adulthood for identification of the presence of transgene by PCR analysis.
Lo, J.H., Chen, T.T. 2010. CCAAT/enhancer binding protein Beta-2 is involved in growth hormone-regulated insulin-like growth factor-II gene expression in the liver of rainbow trout (Oncorhynchus mykiss). Endocrinology. 151:2128-2139.
Chen, M., Chiou, P., Liao, Y., Lin, C., Chen, T.T. 2010. Development and characterization of five rainbow trout pituitary single-cell clone lines capable of producing pituitary hormones. Journal of Endocrinology. 205:69-78.
Sullum, U.W., Chen, T.T. 2010. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri. Marine Biotechnology. DOI: 10.1007/s10126-010-9269-z.