Figure 2
Knocking down heterochromatic gene function by RNAi. (a) RNAi in S2 cells. At 72 h after double-stranded RNA (dsRNA) injection, cells are fixed with methanol:acetic acid and chromosomes are prepared by air drying. Chromosomes of control and treated cells are stained by DAPI, and cytological analysis is performed to reveal apparent mitotic defects. At the bottom of the figure, an aberrant chromosome condensation morphology after RNAi of a 2Rh gene model is shown (right panel). This aberrant phenotype clearly differs from that of the nontreated control cells (left panel). (b) In vivo RNAi using dsRNA-producing transgenes coupled to the GAL4/UAS expression system. In symmetrical transcription, dsRNA synthesis can be induced by cloning, as in the SympUAST vector, a single gene fragment between two convergent arrays of the GAL4-responsive UAS sequences. This strategy permits the use of a long DNA fragment and results in a very stable plasmid both in the bacterial host during the cloning procedure and in the Drosophila genome after transgenesis. dsRNA can also be efficiently produced in vivo by mono-directional transcription of inverted repeats (IR) of a given DNA fragment under the control of a single UAS regulatory region. In this case, the inverted repeats should be separated by a short DNA spacer (100–200 bp) to avoid any rearrangement due to the recombinogenic potential of contiguous IR in the bacterial host and Drosophila genome. The synthesized dsRNA is then processed by the Dicer RNase, which is a component of the RNAi silencing complex (RISC), located in the cytoplasm. The Dicer RNase cleaves the exported dsRNA into smaller, 21-nucleotide small inhibitory RNAs (siRNAs). The siRNAs are used by the RISC complex as a template for destroying the homologous mRNA (in white). Through this mechanism, specific messages can be degraded in a cell-autonomous manner and in specific tissues according to the expression pattern of the GAL4 enhancer trap line used.
