OF ANTISENSE RNA CONSTRUCTS
Rather than using antisense
oligonucleotides, antisense RNA can be transcribed from a vector that has been
inserted into the cell. First, the target gene is cloned in reverse orientation
so that antisense RNA is produced instead of sense mRNA (see Fig. 5.3B). This
method is believed to inactivate the cellular target mRNA by forming a
heteroduplex of sense/antisense RNA. Heteroduplex formation relies on both RNAs
to unfold. If either RNA has a very stable secondary or tertiary structure,
then the construct may not work inside the cell.
The advantage of internal
synthesis of antisense RNA is that the antisense expression can be controlled.
If the antisense gene is cloned behind an inducible promoter, then the
antisense RNA is not made until the gene is induced by specific signals or
conditions. This may be useful to allow organ-specific expression of an
antisense gene. Another advantage is that the antisense RNA may be continuously
expressed internally over a long-term period. This avoids the inconvenience and
expense of constant administration of external antisense oligonucleotides.
A target gene can be cloned in the inverse
direction to create an antisense vector. When this is expressed in the cell,
the antisense mRNA and endogenous mRNA bind, thus preventing expression into