FIGURE 4 | Genetic screens for cell-death regulators in Drosophila melanogaster.

a | In an F₂ screen, animals homozygous for a mutation (the red and yellow asterisk), generated through the series of crosses indicated, are examined for phenotypes suggestive of defects in cell death. These might include embryonic lethality or, if the homozygotes are viable, phenotypes such as defective adult structures (eyes or wings), male or female sterility or a short lifespan. A balancer chromosome (Balancer) prevents recombination with the chromosome that is being mutagenized and homozygosed. b | In a clone-based screen, the goal is to generate marked patches of homozygous mutant tissue in an otherwise heterozygous (and therefore viable) background. In brief, the FLP/FRT system is used to drive MITOTIC RECOMBINATION on a specific chromosome arm (the one that carries FRT sites that are targets for the FLP recombinase) in specific populations of cells (those that are mitotically active and that express the FLP recombinase). If these flies are heterozygous for a mutation (as would be the case if one of the parents were mutagenized in the previous generation), then clones of tissue homozygous for the mutation will be generated in the tissue of interest. In the flies illustrated, clones of mutant tissue are generated in the wing for a mutation that blocks normal cell death; expression of green fluorescent protein (GFP) is lost from the wing cells that die (see main text) so the adults contain patches of green (GFP positive) tissue on a dark (heterozygous or homozygous wild-type (the twin-spot)) background. c | In a dominant modifier screen, wild-type flies are mutagenized and crossed to flies that are in some way sensitized. In this case, they have small eyes owing to expression of a death activator in the eye. Progeny are screened directly for enhancers or suppressors, which might encode other pathway components. An important limitation of the dominant modifier screen is that only components for which activity is rate-limiting (sensitive to a two fold decrease or overexpression) in the sensitized background will be identified. Therefore, to gain a complete picture of a pathway it is often necessary to carry out many screens using flies that are sensitized at different points in a pathway (reviewed in Ref. 19). d | In a cell-culture-based RNA interference (RNAi) screen, cells in 96-well plates are treated with dsRNA for many genes (as many as you want to test) and some cellular phenotype is assayed several days later. In the illustrated case, the cells have been treated with a mild death stimulus that causes a fraction of them to die (the pink wells). Inactivation of genes that promote cell death results in increased cell survival (green wells), whereas inactivation of genes that promote cell survival result in increased cell death (red wells). EMS, ethyl methanesulphonate; M, mitosis.

Download file

If the slide opens in your browser, select "File > Save As" to save it.