Mouse Model for the Human AGAT Deficiency: A Pathopsychological Model of Isolated Creatine Deficiency

Creatine is an essential component of cellular energy storage and utilization in tissues with high energy requirements such as muscle, and brain. In the creatine pathway two main categories of disorders are currently recognized: disorders in creatine biosynthesis (AGAT and GAMT), and disorders of cellular creatine transport (CRTR). To determine the role of an isolated and absolute creatine deficiency due to the recently new discovered enzyme defect in creatine synthesis, the arginine:glycine amidinotransferase (AGAT) deficiency, we purpose to generate a traditional knockout mouse model lacking this gene. This will include the following steps: 1. Development of a targeting construct in order to replace the gene of interest with one that is altered and results in non-functional AGAT gene. 2. Selection of ES cells with homologous recombination of non-functional AGAT gene. 3. Microinjection of AGAT knock out ES cells into blastocysts and pseudopregnant recipient female mice and germ line transmission of targeted gene. 4. Breeding studies & further characterization of the phenotype. By positioning a promoterless ß-galactosidase-neomycin resistance fusion gene (ß-geo) down stream of the AGAT promoter we can both select for homologous recombination and monitor AGAT gene expression in the resulting heterozygous embryos by X-gal staining. The splice acceptor site upstream of the ß-geo fusion gene should lead to splicing from exon 1 of AGAT to ß-geo, generating a stable transcript that confers drug resistance.We plan to carry out investigations into the neuroprotective role of creatine in the brain tumorigenic process, and the neuroprotective role of creatine in radiation and asphyxia induced tissue damage using the AGAT mouse model. Longterm goal is to reduce the deleterious side effects on CNS tissue during radiation by using the neuroprotective effects of ceatine administration.