RNAi is frequently used to inactivate genes in Drosophila and mammalian cell culture lines. Indeed, a set of 15,000 Drosophila RNAi molecules (one for every coding gene) allows researchers, in several months, to test the role of every fly gene in any process that can be monitored using cultured cells. RNAi has also been widely used to study gene function in whole organisms, including the nematode pool of barcoded yeast mutants, each deleted for a different gene
E. coli, expressing double-stranded RNA, eaten by worm
Figure 8–58 Gene function can be tested by RNA interference. (A) Double-stranded RNA (dsRNA) can be introduced into C. elegans by (1) feeding the worms E. coli that express the dsRNA or (2) injecting the dsRNA directly into the animal’s gut. (B) In a wild-type worm embryo, the egg and sperm pronuclei (red arrowheads) come together in the posterior half of the embryo shortly after fertilization. (C) In an embryo in which a particular gene has been inactivated by RNAi, the pronuclei fail to migrate. This experiment revealed an important but previously unknown function of this gene in embryonic development. (B and C, from P. Gönczy et al., Nature 408:331–336, 2000. With permission from Macmillan Publishers Ltd.)
C. elegans. When working with worms, introducing the double-stranded RNA is quite simple: the RNA can be injected directly into the intestine of the animal, or the worm can be fed with E. coli engineered to produce the RNA (Figure 8–58). The RNA is amplified (see p. 431) and distributed throughout the body of the worm, where it inhibits expression of the target gene in different tissue types. RNAi is being used to help in assigning functions to the entire complement of worm genes (Figure 8–59).
A related technique has also been applied to mice. In this case, the RNAi molecules are not injected or fed to the mouse; rather, recombinant DNA techniques are used to make transgenic animals that express the RNAi under the control of an inducible promoter. Often this is a specially designed RNA that can fold back on itself and, through base-pairing, produce a double-stranded region that is recognized by the RNAi machinery. In the simplest cases, the process inactivates only the genes that exactly match the RNAi sequence. Depending on the inducible promoter used, the RNAi can be produced only in a specified tissue or only at a particular time in development, allowing the functions of the target genes to be analyzed in elaborate detail.
RNAi has made reverse genetics simple and efficient in many organisms, but it has several potential limitations compared with true genetic knockouts. For
C. elegans each well contains WORMS INGEST E. coli; RESULTING PHENOTYPES RECORDED AND ANALYZED
E. coli expressing a different dsRNA ADD TO WELLS IN PLATE 96-well plate