chapter  20
33 Pages

Inhibition of Gene Expression

There are currently a variety of approaches used to silence the expression of endogenous genes. For years it has been demonstrated that gene expression can be suppressed by either synthetic antisense oligonucleotides or by expression of the antisense orientation of the gene of interest.1-4 Although the detailed mechanisms of antisense deoxyribonucleic acid/ribonucleic acid (DNA/RNA) inhibition are not well understood, they have been widely applied in eukaryotes in which the antisense oligonucleotides or antisense RNA (asRNA) transcripts inhibit the expression of specific sense RNAs. It is believed that this inhibition is due to the interaction of the antisense sequence with the endogenous complementary or sense RNA transcripts through hydrogen bonding. This interaction is believed to block the processing of translation of the sense RNA, and the duplex of antisense DNA and sense RNA may also be rapidly degraded within the cells. Therefore, the basic principle of using antisense DNA and RNA can be directed toward partially or completely inhibiting the expression of a target gene.2-7

Unfortunately, there are no useful rules or guidelines for engineering effective antisense constructs. Consequently, antisense gene silencing tends to be an unpredictable technology,8 and there is little current research directed at understanding and improving the use of antisense gene silencing. Three limitations are commonly encountered with antisense gene silencing: (1) antisense constructs often silence the target gene in only a small minority (10 to 30%) of transformants; (2) antisense constructs typically cause only partial gene silencing, i.e., they produce “leaky” phenotypes; and (3) antisense gene silencing often produces epigenetically unstable phenotypes, necessitating extensive screening and vigilant monitoring to identify and maintain stable lines of transformants. Leaky phenotypes, for example, are thought to be produced when antisense RNA does not accumulate to levels sufficient to interfere with all target transcripts in the appropriate cellular compartments and cell types. Another possible cause of partial silencing is that many antisense RNAs do not always pair efficiently with the target RNA due to interference by secondary structures in the asRNA and/or the target RNA.9,10 Despite these potential difficulties, antisense technology has been profoundly useful in elucidating gene functions when the proper attention to detail is maintained.