2010 Annual Report
1a.Objectives (from AD-416)
The objectives of this cooperative research project are to:.
1)Complete the development of rainbow trout breeding stocks that consist of true-breeding individuals for the cecropin gene, a gene which increases disease resistance;.
2)Complete the identification and characterization of genes in rainbow trout macrophage cells that are responsive to induction by cecropin B, and;.
3)Develop rainbow trout broodstock for aquaculture with enhanced muscle growth by down-regulating the MSTN-1 gene.
1b.Approach (from AD-416)
Bacterial and viral pathogens will be used to challenge F2 transgenic fish and those that exhibit resistance to one or more of the pathogens will be bred together as the initial stages for development of a true-breeding stock. In addition, the production of gynogens from the eggs of resistant animals will be attempted to shorten the time to make the genes completely homozygous. Rainbow trout macrophages will be treated with cecropins and the expression of genes will be monitored by the use of cDNA subtraction hybridization. This technique will allow the identification of the genes which play a role in the adaptive immune response in rainbow trout. Finally, the development of rainbow trout broodstock with increased muscle growth will be attempted by use of the RNAi gene constructs to down-regulate the MSTN gene. Preliminary results suggest that this approach will lead to strains with enhanced somatic growth rate, reducing the costs for care, feeding and rearing space.
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.
The ADODR is in frequent contact with the cooperator through phone calls, email, and site visits in addition to receipt of written reports.