Mouse ataxin-3 functional knock-out model.

Area of Science:

• Genetics
• Neuroscience
• Molecular Biology

Background:

• Spinocerebellar ataxia 3 (SCA3) is a genetic neurodegenerative disorder caused by expanded CAG repeats in the ATXN3 gene.
• The precise pathogenic mechanisms of SCA3 remain incompletely understood.
• Existing transgenic mouse models do not fully recapitulate human SCA3, highlighting the need for a humanized knock-in model.

Purpose of the Study:

• To generate a humanized mouse model for Spinocerebellar ataxia 3 (SCA3) using a knock-in strategy.
• To introduce human ATXN3 cDNA with expanded CAG repeats into the mouse genome.
• To investigate the pathogenesis of SCA3 in a humanized genetic context.

Main Methods:

• Cloning human ATXN3 cDNA with 69 CAG repeats from an SCA3 patient.
• Utilizing a knock-in strategy to insert the human transgene into the mouse ATXN3 locus.
• Employing western blot, immunohistochemistry, and RNA expression analysis to characterize the resulting mice.

Main Results:

• The knock-in strategy resulted in mice with functional ataxin-3 knock-out properties, lacking protein expression.
• Alternative splicing events were observed, producing mRNA isoforms from human and mouse exons.
• A very low level of the intended correct splice isoform was detected, suggesting splicing aberrations.
• Bioinformatic analysis indicated that splicing site alterations, not inherent site strength, likely caused aberrant splicing.

Conclusions:

• The study successfully generated a viable and fertile ataxin-3 knock-out mouse model.
• Unexpected splicing defects occurred, preventing the expression of the humanized SCA3 transgene.
• The findings offer valuable insights into the complex splicing of the Atxn3 gene.
• This research provides crucial information for future attempts to develop accurate knock-in SCA3 models.