Go Back

Double-Stranded RNA Interference with Gene Expression: RNAi

A surprising discovery has recently revolutionized C. elegans "reverse genetics"-the process by which scientists begin with a known gene sequence and attempt to define its biological function by disrupting its activity in vivo. The finding is that introduction of a double-stranded RNA (dsRNA) including coding sequences of almost any known gene can specifically disrupt the function of that gene in vivo. Introduction of dsRNA mimics a gene knockout (for example, by deletion of the gene itself). However, the resulting effects on an animal are referred to as a phenocopy since it copies the phenotype of a loss-of-function mutation of that gene, but is not really inherited like a true gene deletion would be.

RNA is easily synthesized in a test tube by adding phage RNA polymerases that recognize phage promoters housed in expression vectors (of course rNTPs are required as well). Phage T7 and T3 promoters are included in many expression vectors to enable transcription of individual strands of the gene when T7 or T3 polymerases are provided. Often both T7 and T3 promoters flank the cloned gene so that use of T7 polymerase can drive expression of one strand and T3 can drive expression of the complementary strand (scheme below).

RNAi has some remarkable properties: