Gene synthesis

Plasmids for transfection were obtained as previously described.²² (link) In short, PCR products for cloning were generated with primers flanking the full-length ATXN3 transcript (see Table 2) using AON transfected fibroblast cDNA as template. Full-length or exon 10-skipped products were gel extracted, purified, and ligated in the pGEM-T Easy Vector (Promega) using the 5′-A overhangs. Mutations of the UIMs and deletion of cysteine 14 was generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) as described previously using primers containing the mutation (Table 2).⁴⁰ (link) Expanded ataxin-3 with 71Q was obtained by gene synthesis (GenScript), a mixture of CAG and CAA codons was generated to improve stability during the cloning process. Constructs were then subcloned into the pAcGFP-C1 vector (Clontech) using notI digestion, resulting in an N-terminally GFP-tagged ataxin-3 protein expression. The mCherry-LacR-RNF8 construct has been described previously.²¹ (link) Purified ataxin-3 proteins were produced using the pET28a vector (Merck Millipore) and BL21 E. coli (New England Biolabs) as previously described.²² (link) All constructs were verified using Sanger sequencing.