title: Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors.
abstract: Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction.
authors: National Muscular Dystrophy Research Centre, Howard Florey Institute, Fitzroy, Victoria, Australia.
authors: Marian
authors: M
authors: Todaro
authors: Anita
authors: A
authors: Quigley
authors: Magdalena
authors: M
authors: Kita
authors: Judy
authors: J
authors: Chin
authors: Kym
authors: K
authors: Lowes
authors: Andrew J
authors: AJ
authors: Kornberg
authors: Mark J
authors: MJ
authors: Cook
authors: Robert
authors: R
authors: Kapsa
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016189:Dystrophin / Q000235:genetics*; D005189:False Positive Reactions; D008297:Male; D051379:Mice; D018101:Mice, Inbred mdx; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA / Q000379:methods*; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection
chemical_list: D016189:Dystrophin