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Nucleic Acids Research, 2002, Vol. 30, No. 11 2329-2339
© 2002 Oxford University Press

Analysis of inhibitory action of modified U1 snRNAs on target gene expression: discrimination of two RNA targets differing by a 1 bp mismatch

Peng Liu, Amy Gucwa, Mary Louise Stover, Emily Buck, Alexander Lichtler and David Rowe*

Department of Genetics and Developmental Biology, Mail Code 3301, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA

The modified U1 snRNA gene can suppress expression of a target transgene. In the present study, its potential utility to inhibit a dominant negative/gain of function mutation is explored. Using a green fluorescent protein (GFP) target gene, inhibition was achieved in all cells transduced with U1antiGFP directed at multiple sites within GFP. Using a chloramphenicol acetyltransferase (CAT) target gene, inhibition was not increased by increasing the hybridization domain from 10 to 16 bp or when a site in an upstream exon or intron was targeted. To determine if a U1 anti-target design could discriminate between two transcripts that differ by a 1–2 bp mismatch, GFPtpz and GFPsaph were chosen as targets because they share sequence homology except for three regions where a 1, 2 or 3 bp mismatch exists. The results demonstrated that U1antiGFP correctly reduced its cognate GFP expression by >90% and therefore U1 anti-target constructs are able to discriminate a 1 or 2 bp mismatch in their target mRNA. Thus, these U1 anti-target constructs may be effective in a strategy of somatic gene therapy for a dominant negative/gain of function mutation due to the discreteness of its discrimination. It may complement other anti-target strategies to reduce the cellular load of a mutant transcript.

* To whom correspondence should be addressed. Tel: +1 860 679 2324; Fax: +1 860 679 8345; Email: drowe{at}nso1.uchc.edu


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