title: Markerless modification of trinucleotide repeat loci in BACs.
abstract: Transcription and splicing of human genes are regulated by nucleotide sequences encoded across large segments of our genome, and trinucleotide repeat expansion mutations can have both profound and subtle effects on these processes. In the course of our work to understand the impact of the Spinocerebellar Ataxia type 8 (SCA8) CTG repeat expansion on the transcription and splicing of the RNAs encoded near the SCA8 locus, we have developed a set of reagents and protocols for modifying large genomic BAC clones of this region. We describe the two-step procedure that allows us to precisely replace unexpanded trinucleotide repeats with expanded variants of these repeat sequences without leaving any exogenous sequences in the final constructs, and we discuss how this approach can be adapted to make other desired sequence changes to these genomic clones.
authors.affiliation: Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Kellie A
authors.initials: KA
authors.lastname: Benzow
authors.forename: Michael D
authors.initials: MD
authors.lastname: Koob
mesh_terms: D001089:Arabinose / Q000494:pharmacology; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D018274:Electroporation; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic; D018911:Trinucleotide Repeats / Q000235:genetics*
chemical_list: D009419:Nerve Tissue Proteins; D004247:DNA; D001089:Arabinose