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:
- RNAi is highly gene specific. The dsRNA should include at least about 300 bp of coding sequence homology, although specific requirements for size and amount of coding sequence that must be present are not well understood. It does work if introns are present in the intended interfering RNA. It is not clear how susceptible one member of a given gene family is to the inhibitory effects of a dsRNA of another closely related family member, but it has been suggested on the basis of some data that genes 80% identical at the nucleotide level are spared inhibitory effects of their closely related family member.
- dsRNA appears to move freely within the worm. It is not necessary to inject dsRNA directly into the gonad to get progeny that exhibit RNAi effects. Injection of dsRNA into the tail or gut will do! This indicates that the introduced dsRNA species must be able to move across cell boundaries freely.
Even more remarkably, nematodes can be soaked in dsRNA or can be fed plasmids that make dsRNA and consequently exhibit RNAi effects. Although this protocol is not particularly potent, we will give it a try to look for RNAi effects on some of the genes you choose to focus on for your 315 projects.
- The mechanism of RNAi is not well understood. Some data suggests that cells have such a response to protect themselves against the invasion of foreign nucleic acids or to protect them selves against transposition and viruses.
- RNAi works in planaria, trypanosomes, flies, mice and plants. This suggests that it might be a widespread phenomenon and might be used in the future for gene inactivation in humans!