title: Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference.
abstract: Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125I-alpha-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of alphaS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders.
authors.affiliation: Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, UK.
authors.forename: Amr
authors.initials: A
authors.lastname: Abdelgany
authors.forename: Matthew
authors.initials: M
authors.lastname: Wood
authors.forename: David
authors.initials: D
authors.lastname: Beeson
mesh_terms: D000483:Alleles; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D015536:Down-Regulation; D020868:Gene Silencing; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011950:Receptors, Cholinergic / Q000235:genetics*; D013569:Synapses; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D009841:Oligonucleotides; D034741:RNA, Small Interfering; D011950:Receptors, Cholinergic; D012313:RNA; D004247:DNA