Nucleic Acids Research, Vol 25, Issue 16 3218-3227, Copyright © 1997 by Oxford University Press
HM Engdahl, TA Hjalt and EG Wagner
This communication describes a two unit antisense RNA cassette system for
use in gene silencing. Cassettes consist of a recognition unit and an
inhibitory unit which are transcribed into a single RNA that carries
sequences of non-contiguous complementarity to the chosen target RNA. The
recognition unit is designed as a stem-loop for rapid formation of long-
lived binding intermediates with target sequences and resembles the major
stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit
consists of either a sequence complementary to a ribosome binding site or
of a hairpin ribozyme targeted at a site within the chosen mRNA. The
contributions of the individual units to inhibition was assessed using the
lacI gene as a target. All possible combinations of recognition and
inhibitory units were tested in either orientation. In general, inhibition
of lacI expression was relatively low. Fifty per cent inhibition was
obtained with the most effective of the constructs, carrying the
recognition stem-loop in the antisense orientation and the inhibitory unit
with an anti-RBS sequence. Several experiments were performed to assess
activities of the RNAs in vitro and in vivo : antisense RNA binding assays,
cleavage assays, secondary structure analysis as well as Northern blotting
and primer extension analysis of antisense and target RNAs. The problems
associated with this antisense RNA approach as well as its potential are
discussed with respect to possible optimization strategies.
ARTICLES
A two unit antisense RNA cassette test system for silencing of target genes
Department of Microbiology, Swedish University of Agricultural Sciences, Genetic Center, Box 7025, Genetikvagen 1, S-75007 Uppsala, Sweden. engdahl@mikrob.slu.se
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