2009 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, s reducing the costs for care, feeding and rearing space.
We have conducted challenge studies on transgenic fish with the bacterial pathogen A. salmonicida and the viral pathogen IHNV. Results of the challenge studies showed fish of these transgenic families consistently exhibited resistant characteristics to A. salmonicida and IHNV. Sperm samples from these families have been cryopreserved. Furthermore, we initiated work to breed fish homozygous for the transgene by androgenesis. To date, we have successfully bred two cecropin P1 transgenic fish families; although androgenesis has also been conducted on the remaining 7 cecropin P1 transgenic fish families, the resulting fish are too small to be sampled for genotyping at this moment. Fin tissue samples will be collected from these fish for genotyping by this fall and we expect to complete genotyping these fish by December 2009.
The ADODR is in frequent contact with the cooperator through telephone calls, email, and annual site visits in addition to receipt of written reports.
Chen, T.T., Chen, M.J., Chiou, T., Lu, J.K. 2009. Transfer of foreign DNA into aquatic animals by electroporation. In: Nakamura,H., editor. Electroporation and Sonoporation in Developmental Biology. Tokyo, Japan: Springer. p. 229-237.
Chiou, P.P., Khoo, J., Chun, C., Chen, T.T. 2005. Transgenic Fish. In: Meyers, R., editor. Encyclopedia of Molecular Cell Biology and Molecular Medicine. 2nd Edition, Vol.14. Federal Republic of Germany: VCH Verlagsgesellschaft mbH. p. 473-503.
Lo, J.H., Chiou, P.P., Chen, T.T. 2007. Molecular cloning and expression analysis of rainbow trout(Oncorhynchus mykiss)CCAAT/enhancer binding protein genes and their responses to induction by GH in vitro and in vivo. Journal of Endocrinology. 194:393-406.
Sallum, U.W., Chen, T.T. 2008. Inducible Resistance of Fish Bacterial Pathogens to the Antimicrobial Peptide Cecropin B. Antimicrobial Agents and Chemotherapy. 52(9):3006-3012.
Chiou, P.P., Lin, C., Bols, N.C., Chen, T.T. 2007. Characterization of virus/double-stranded RNA-dependent induction of antimicrobial peptide hepcidin in trout macrophages. Developmental and Comparative Immunology. 31:1297-1309.
Chen Hung-Chih, M., Li, Y., Chang, Y., Wu, S., Gong, H., Lin, G., Chen, T.T., Wu, J. 2006. Co-induction of hepatic IGF-I and progranulin mRNA by growth hormone in tilapia, Oreochromis mossambiccus. General and Comparative Endocrinology. 150:212-218.
Chiou, P.P., Khoo, J., Bols, N.C., Douglas, S., Chen, T.T. 2005. Effects of linear cationic x-helical antimicrobial peptides on immune-relevant genes in trout macrophages. Developmental and Comparative Immunology. 30:797-806.