In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis

Production of mouse models of inherited neurodegenerative diseases is an im portant step towards understanding the mechanism of neurotoxicity and for t esting potential therapies. We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disord er caused by expansion of a CAG repeat within the androgen receptor (AR) ge ne. To permit generation of mice that will show a SBMA phenotype within the ir life span, we decided to obtain a yeast artificial chromosome (YAC) carr ying the AR gene and introduce CAG repeat mutations numbering 100 or more t riplets. SBMA patients with more than 70 CAGs have never been observed; the refore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia cot i. Although we set out to expand this repeat tract using a recombination pa radigm involving two plasmid co-propagation, we did not observe large expan sions. We were instead able to incrementally generate repeat tracts from 10 0 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability. In the course of our experiments that yielded thes e CAG repeat tracts, we evaluated the role of repeat orientation, vector co propagation, and recA function on the expansion process. We then used one o f the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats. AR YAC CAG100 will serve as a valuable reagent f or the production of a SBMA mouse. (C) 2000 Elsevier Science B.V. All right s reserved.