In Vitro Expansion of CAG, CAA, and Mixed CAG/CAA Repeats.
Polyglutamine diseases, including Huntington's disease and a number of spinocerebellar ataxias, are caused by expanded CAG repeats that are located in translated sequences of individual, functionally-unrelated genes. Only mutant proteins containing polyglutamine expansions have long been thought to be pathogenic, but recent evidence has implicated mutant transcripts containing long CAG repeats in pathogenic processes. The presence of two pathogenic factors prompted us to attempt to distinguish the effects triggered by mutant protein from those caused by mutant RNA in cellular models of polyglutamine diseases. We used the SLIP (Synthesis of Long Iterative Polynucleotide) method to generate plasmids expressing long CAG repeats (forming a hairpin structure), CAA-interrupted CAG repeats (forming multiple unstable hairpins) or pure CAA repeats (not forming any secondary structure). We successfully modified the original SLIP protocol to generate repeats of desired length starting from constructs containing short repeat tracts. We demonstrated that the SLIP method is a time- and cost-effective approach to manipulate the lengths of expanded repeat sequences.
Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704 Poznan, Poland. gfigura@man.poznan.pl.
Grzegorz
G
Figura
Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704 Poznan, Poland. edytak@ibch.poznan.pl.
Edyta
E
Koscianska
Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704 Poznan, Poland. wlodkrzy@ibch.poznan.pl.
Wlodzimierz J
WJ
Krzyzosiak
D000067699:Ataxin-3 / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D019680:Trinucleotide Repeat Expansion; D018911:Trinucleotide Repeats
D000067699:Ataxin-3
SLIP; in vitro expansion; plasmid constructs; polyglutamine diseases; repeats in vitro cloning; trinucleotide repeats