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.
With the rapid growth of the human population in the world and the increased consumption of fishery products for health reasons, the world demand of fishery products is mounting rapidly. To this, many countries have turned to various forms of intensive aquaculture. However, the rapid escalation of fish production by aquaculture has resulted in several negative impacts such as disease outbreaks in aquaculture facilities. These negative impacts have brought about significant economic losses in the aquaculture industry. A solution to some of these problems is the development of disease resistant fish species by genetic engineering. This project focused on development of rainbow trout strains that are resistant to infection by bacterial and viral pathogens by introducing antimicrobial peptide genes through genetic engineering. Transgenes containing pig cecropin P1 and synthetic cecropin-like (CF-17) genes were introduced into rainbow trout via a sperm mediated gene transfer method. The P1 generation transgenic animals were crossed with non-transgenic fish to establish F1 founder transgenic families. Totally 20 families of cecropin P1 and 10 families of CF-17 transgenic fish were established. Each of these transgenic families was crossed to non-transgenic fish to generate F2, F3 and F4 transgenic offspring and the fingerlings of these fish were challenged with Aeromonas salmonicida (an important bacterial pathogen for rainbow trout) and infectious hematopoietic necroses virus (IHNV). Repeated challenge studies revealed that a total of nine families of cecropin P1 transgenic fish and five families of CF-17 transgenic fish exhibited the characteristic of resistance to Aeromonas salmonicida and IHNV. Each of these families was bred into all male homozygous state and the sperm samples from all families were cryo-preserved. Although we have attempted to produce rainbow trout strains by introducing siRNA transgenes to down-regulate the expression of myostatin gene many times, we failed to produce any viable transgenic trout.
The ADODR is in frequent contact with the cooperator through phone calls, email, and site visits in addition to receipt of written reports.