Insertion of expanded CAG trinucleotide repeat motifs into a yeast artificial chromosome containing the human Machado-Joseph disease gene

Machado-Joseph disease or spinocerebellar ataxia 3 (SCA3) is a progressive neurodegenerative disorder caused by pathological expansion of a trinucleot ide repeat motif present within exon 4 of the MJD1 gene. Previous attempts to create a transgenic animal model have failed to produce a neurological d eficit truly representative of the disease phenotype. This appears to be th e result of inappropriate expression of the mutant protein in neuronal popu lations generally spared in the disease state. Introduction of a human dise ase gene in the context of a yeast artificial chromosome clone containing e ndogenous regulatory elements would enhance the potential for correct tissu e/cell-specific expression at physiological levels. We report the introduct ion of expanded CAG repeat motifs into a 250 kb yeast artificial chromosome clone spanning the MJD1 locus using two rounds of homologous recombination , Transformants exhibited both expansions and contractions of the motif wit h alleles ranging in size from 48 to 84 repeat units. The availability of t hese clones for modelling of the disease in transgenic animals should allow elucidation of the role of repeat length in the phenotypic spectrum of the disease. (C) 1999 Elsevier Science B.V. All rights reserved.