In spite of the remarkable success in the use of Drosophila as a model organism, a major disadvantage for workers in the field is the lack of a method of targeted gene disruption. Dr. Kent Golic proposes to develop two strategies for making gene knockouts that can be transmitted through the Drosophila germline. Both of these strategies take advantage of his expertise in utilizing FLP-mediated recombination in the fly. In the first of these strategies, the induction of FLP recombinase from a heat shock promoter will drive the excision as a circle of DNA sequences located between two FRT sites on a construct previously inserted into the genome. In some rare cases, this circle will then reintegrate into the genome by homologous recombination at sites where the genome contains target gene sequences related to those in the circle. In other cases, reintegration may be by """"""""parahomologous targeting"""""""" in which chromosome rearrangements at the target gene accompany the integration event. The challenge then is to select for these rare reintegration events, which Dr. Golic intends to do with a novel strategy in which the excision of the circle will simultaneously inactivate a dominant male-sterile form of the beta2 tubulin gene and reconstruct a wild-type version of the same gene. As a result, male flies that were originally sterile should be able to make functional sperm in the descendants of germline stem cells where the excision occurred and where the wild-type beta2 tubulin gene was subsequently retained by virtue of its reintegration. Any fertile males are thus good candidates to have disruptions of the targeted gene; this will subsequently be verified by molecular techniques. In the second strategy, Dr. Golic will attempt to excise a linear fragment of DNA containing target gene sequences. The rationale is that the linear fragment should be more recombinogenic that circular DNA. Two different variants of this strategy will be tried, though both eventually will use the beta2 tubulin selection described above to identify animals in which the linear fragment recombined back into the genome. First, DNA located between two FRT sites will be excised as a circle by FLP recombinase as before. This circle will also contain a site for a rare-cutting endonuclease such as I-SceI. If expression of the nuclease is also under heat shock control, then the circle will be linearized. Alternatively, Dr. Golic will attempt to cut out the linear piece between two FRT sites by using FLP to make double strand breaks at the FRTs. This will require the identification of a mutant form of FLP that cuts at FRTs but does not recombine them. Dr. Golic describes a selection scheme in yeast to find such mutant forms of FLP, looking for altered FLPs that stimulate gene conversion at FRT by making double strand breaks.
| Rong, Y S; Golic, K G (2000) Gene targeting by homologous recombination in Drosophila. Science 288:2013-8 |
