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ABSTRACT
Following the advent of a gene targeting technique in Drosophila, different methods have been developed to modify the Drosophila genome. The initial demonstration of gene targeting in flies used an ends-in method, which generates a duplication of the target locus. The duplicated locus can then be efficiently reduced to a single copy by generating a double-strand break between the duplicated segments. This method has been used to knock out target genes by introducing point mutations. A derivative of this method is reported here. By using different homologous regions for the targeting and reduction steps, a complete deletion of the target gene can be generated to produce a definitive null allele. The breakpoints of the deletion can be precisely controlled. Unlike ends-out targeting, this method does not leave exogenous sequence at the deleted locus. Three endogenous genes, Sir2, Sirt2, and p53 have been successfully deleted using this method.
TWO general strategies can be used to modify a gene of interest by homologous recombination. Either an ends-in or an ends-out targeting strategy is chosen on the basis of the desired outcome. These terms refer to the placement of a DNA double-strand break (DSB) that is used to stimulate recombination. For ends-in targeting, a double-strand break is generated within the target-homologous region of the donor construct. Recombination between the donor and target typically generates a duplication of the target locus. In spite of this duplication, ends-in donors can be designed in several ways to generate mutant alleles of the target locus. Homologous recombination can be achieved using just a fragment of the target gene to generate truncated gene copies. Alternatively, point mutations may be introduced into each of the two copies, generating two mutant copies of the gene. Finally, in a two-step process, a single-copy mutant allele may be generated. All these methods have been used to produce mutants in Drosophila (RONG and GOLIC 2000, 2001; RONG et al. 2002; SEUM et al. 2002; DOLEZAL et al. 2003; EGLI et al. 2003; ELMORE et al. 2003; LANKENAU et al. 2003; LIU and KUBLI 2003; SOGAME et al. 2003). One problem with these methods is that, without additional characterization, it is difficult to be absolutely sure that a null allele has been generated.
A deletion of a...