Silence of the genes.

T he genomes of many species, including man, have been laid bare with hundreds of known and thousands of unknown genes. A major challenge in present day biology is to decipher the molecular function of the unknown genes. Gene knockout by homologous recombination has proven to be very useful but is laborious and expensive. Antisense and ribozyme technology have been useful but are not always reliable or robust. RNA interference has emerged as a novel pathway that offers great hope and promise to study the functions of a vast number of genes. The advent of this technology could not have come at a better time. Briefly, in invertebrates, long double-stranded RNA molecules are processed by the endonuclease dicer into 21to 23-nt small interfering RNAs (siRNAs), which are then incorporated into RNA-induced silencing complex, a multicomponent nuclease complex that selects and degrades mRNAs that are homologous to the initially delivered double-stranded RNA (1, 2). In mammalian systems, introduction of long double-stranded RNA ( 50 bp) results in systemic, nonspecific inhibition of translation due to activation of the PKR response. This formidable obstacle can be overcome by the use of synthetic siRNAs ( 30 bp) that can be either delivered exogenously (3) or expressed endogenously from RNA polymerase III promoters, resulting in a powerful tool for achieving specific down-regulation of target mRNAs (4–8). Currently, in mammalian systems, the only way to generate a whole-genome knockdown screening by using RNA interference is to systematically generate synthetic or polymerase III-transcribed siRNA hairpins for every known target gene. Because only 25% of selected target siRNA sequences are functional, several synthetic siRNAs need to be generated and tested for every target gene. Algorithms are being developed to predict effective siRNA sequences for efficient screening of large numbers of genes, but presently they have not been validated and may require automation (9).