The long-term objective of this research is to develop strains of mice with altered or deleted cystathionine beta-synthase (CBS) gene(s) to model homocystinuria and premature peripheral and cerebrovascular disease (PPAD/CeVD). CBS is a central enzyme at the transsulfuration/remethylation branch point of sulfur amino acid metabolism. It is a tetramer of 63 kDa subunits. Its gene resides on human chromosome 21 and mouse chromosome 17. Inherited CBS deficiency precipitates the most common form of homocystinuria; this condition is now being diagnosed more frequently than PKU in some American states with neonatal screening programs. Homocystinuria is accompanied by mental retardation, grand mal seizures, dislocated optic lenses, skeletal disorders, and fatal vascular episodes. About 1/3 of unselected patients with PPAD/CeAD are reported to be heterozygous for CBS deficiency. There are currently no animal models for these conditions. Recent advances in gene excision via homologous recombination in embryonic stem cells together with our studies of the sequence and organization of CBS genes will allow us to develop animals deficient in one or both cbs alleles. Our specific aims are: 1) To evaluate the mouse CBS gene to facilitate molecular cloning of the targeting sequence; 2) to construct such a mouse CBS targeting sequence which will excise approximately half of the mouse CBS gene; 3) to prepare ES cells deficient in one CBS allele by homologous recombination with this sequence, and to use these cells to generate hetero- and homozygous mice; 4) to examine the pathophysiology and biochemistry of homozygous CBS deficiency in animal model; 5) to determine whether half-normal CBS gene dosage is associated with PPAD/CeVD in the animal model; and 6) to replace the transcribed portion of the mouse CBS gene with normal and mutant human CBS cDNA, and to evaluate the effects of human mutations on the animal. Cellular, molecular, biochemical, anatomical, and developmental approaches will be utilized including: i) Gene excision by homologous recombination in embryonic stem cells; ii) generation of chimeric mice by ES cell blastocyst colonization; iii) breeding of the heterozygous and homozygous animal models; iv) evaluation of CBS gene expression through PCR, nucleic acid and protein blotting as well as enzyme assay; v) gross and microscopic examination of target tissues; vi) neurological, behavioral and developmental analysis of the resulting animals. In addition, the animal models will be made available to other laboratories conducting research on homocystinuria and PPAD/CeVD. These studies will elucidate disturbances of sulfur amino acid metabolism; further, they will permit the correlation of CBS deficiency and the clinical manifestation of disease and should facilitate the development of a biochemical rationale for treatment of certain mutant conditions.
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