Ryan D. McAllister and Dr. David Kooyman, Animal Science
Threonine is one of the 20 common amino acids found in nature. It is a limiting factor for the growth of many non-ruminant animals such as swine and mice and must be obtained from their diet. When threonine is absent from the diet of these animals, the other amino acids present cannot be fully utilized. The result is poor conversion of plant to animal protein (1). We proposed producing transgenic mice using the genes from the known five-step bacterial pathway for threonine synthesis (2). The introduction of these bacterial pathways to mice would eliminate the need for dietary supplementation of threonine. When successful, this model will be used by the USDA to produce threonine independent swine.
The work was completed between January through April 1999. The preliminary steps of the process began by engineering a bacterial plasmid (ring of DNA) containing the desired threonine genes. The various steps of the plasmid production included digesting (cutting) the DNA with restriction enzymes and ligating (pasting) those existing DNA fragments of the threonine pathway to make the template plasmid. This proved to be more difficult than expected.
While working on the project, the DNA fragments of the plasmid were not properly ligating. After much trial and error, we determined that the DNA with which we had been supplied was not correct for our application. Obviously, the production of the plasmid was hampered.
The research will not end once the production of the threonine genetic pathway containing plasmid is completed. With hope, the plasmid will be completed during the summer months of 1999. The correctly engineered plasmid then must be transformed to competent bacterial cells. The transformed cells will then amplify the plasmid. Only after harvesting the new plasmids from the bacteria, will the plasmid DNA be microinjected into mouse zygotes. Finally, the expected expression of the transferred genes will be examined by dietary analysis of the mice.
References
- Rees, W. D., Flint, H. J. and Fuller, M.F. 1990. A Molecular Biological Approach to Reducing Dietary Amino Acid Needs. Bio/Technology 8:629-633.
- Rees, W. D. and Hay, S. M. 1995. The Biosynthesis of Threonine by Mammalian cells: Expression of a Complete Bacterial Biosynthetic Pathway in an Animal Cell. Biochem. J. 309:999-1007.