Gene suppression through RNA interference occurs at the posttranscriptionl level and involves mRNA degradation (Montgomery et al., 1998; Ngo et al., 1998). In addition to playing a powerful role in creating loss-of-function mutants, it probably also plays an essential role in protecting the genome against instability caused by exogenous RNAs (eg. Viruses) (Kasschau et al., 1998) and accumulation of transposons and repetitive sequences (Ketting et al., 1999; Tabara et al., 1999; Hannnon GJ, 2002). Thus the vast nature of RNA interference-like processes may encompass not only gene silencing phenomena but also cellular programs for regulation of genes, inhibition of transposon mobilization, and anti-viral mechanism in plants.
RNAi occurs through a series of steps involving the generation of small interfering RNAs (siRNAs) in vivo through the action of a specific RNAaseIII endonuclease Dicer. The resulting siRNAs mediate the degradation of their complementary RNA by association of the siRNA with a nuclease complex to form what is called the RNA-induced silencing Complex (RISC). In the next step, an unwinding of the siRNA occurs which activates RISC. It is the activated RISC that binds to the target mRNA and finally leads to the loss of expression of the gene it coded (Zamore et al., 2000).
The major hurdle to achieving RNAi in mammals was that dsRNAs longer than 30 nucleotides activated defense mechanisms that resulted in non-specific degradation of RNA transcripts and a general shutdown of host cell protein synthesis (Williams, BR 1997). This obstacle was recently overcome by using in vitro synthesized ~21 nucleotide siRNAs to mediate gene-specific suppression in mammalian cells. These siRNAs are long enough to cause gene suppression but not so long to cause interferon response to take place (Elbashir et al., 2001;Calpen et al., 2001). Once these molecules were identified, several DNA vector-based strategies were developed allowing use of RNAi into mammalian cells. |
No worries about RNA degradation 
