Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference.
Hum Mol Genet
; 12(20): 2637-44, 2003 Oct 15.
Article
in En
| MEDLINE
| ID: mdl-12928480
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Receptors, Cholinergic
/
RNA Interference
/
Alleles
Limits:
Animals
/
Humans
Language:
En
Journal:
Hum Mol Genet
Journal subject:
BIOLOGIA MOLECULAR
/
GENETICA MEDICA
Year:
2003
Document type:
Article
Affiliation country: