Our objective is to develop methods for the selective modification of specific metabolic pathways in intact cells and animals, with the long-term goal of using this technology to analyse the relationships between structure, function and regulation of metabolically important enzymes. Specifically, we propose to inhibit the rate of de novo fatty acid biosynthesis in growing quail by inhibiting the expression of the pace-setting enzyme for the de novo synthesis of long-chain fatty acids, acetyl-CoA carboxylase. We propose to isolate a cDNA clone of avian acetyl-CoA carboxylase and use it to construct a chimeric anti-gene which contains the promoter/enhancer sequence of Rous Sarcoma Virus (RSV) and the structural sequence, in reverse orientation, of the cDNA for acetyl-CoA carboxylase. The RSV promoter/enhancer is a well-characterized, strong promoter which can drive expression of heterologous structural genes in chicken cells. The anti-sense RNA will hybridize to the endogenous mRNA and inhibit production of acetyl-CoA carboxylase. The effectiveness of the anti-sense mRNA at inhibiting expression of the endogenous gene will be tested in fibroblasts in culture infected with a retroviral vector containing the anti-gene. If the cell culture experiments are successful, the anti-gene, in retroviral vector, will be used to infect 1-day-old embryos. Birds which harbor the chimeric gene in the germ line will be identified by assaying blood for viral RNA and erythrocytes for integrated viral DNA. New strains will be established by breeding. We will begin analysis of the requirements for effective inhibition of gene expression with anti-genes using our existing, near full-length cDNA clones for chicken malic enzyme. Japanese Quail are small and have a short life cycle. Regulation of lipogenesis appears to be the same in quail and chickens. Use of quail, as opposed to chickens, reduces the space required to grow and breed the transgenic birds, accelerating the breeding program to isolate strains with the chimeric anti-gene in germ line. In principle, the approaches developed in this proposal should be applicable to any avian or mammalian species.
