A generally applicable gene targeting method in Drosophila melanogaster

Yikang S. Rong
The Stowers Institute for Medical Research,
1000 East, 50th Street,
Kansas City, MO 64111
telephone: (816)926-4103
fax: (816)926-2058
email: yir@stowers-institute.org
prestype: Platform
presenter: Yikang S. Rong

YS Rong*, SWA Titen*, M Golic*, H Xie*, KG Golic*, M Bastiani**, PK Bandyopadhyay**, BM Olivera**, M. Brodsky***, GM Rubin***
*: Stowers Institute, **: Department of Biology, University of Utah, ***: Department of MCB, University of Califonia at Berkeley

Since the development of a new gene targeting method in Drosophila (Rong and Golic 2000), we and others have successfully targeted at least nine different loci throughout the genome. This proves that our method should be generally applicable for introducing specific modification to any endogenous locus in Drosophila. It will allow Drosophila researchers to take full advantage of the complete sequences of the genome in functional studies of Drosophila genes.

We showed that the mechanism of gene targeting by homologous recombination (HR) in Drosophila was essentially identical to that of traditional yeast and mouse targeting. The novelty of the Drosophila method lies in the way in which the donor DNA molecule for targeting was introduced into the cells. Instead of direct delivery by electroporation or chemical treatment of cells, the Drosophila donor was generated within the germ cells of intact animals. This was done by first introducing the donor randomly into the genome as a specially constructed P transposable element. Then, a free moving donor molecule was generated from the chromosomal P element by the concert actions of the yeast FLP site-specific recombinase and the yeast I-SceI site-specific DNA endonuclease which were synthesized in Drosophila cells. To accomplish this, flies were produced by crossing that contained a donor P element in the genome, as well as two transgenes expressing FLP and I-SceI respectively. In the germ cells of these flies, FLP excised the donor as an extrachromosomal circle, and I-SceI made a double strand break (DSB) at its recognition site imbedded in the donor circle. The presence of this DSB stimulated HR between the donor and the target leading to donor integration at the endogenous locus. The end result was a tandem duplication of the target gene. Targeting events were recovered as progeny of these flies after they were mated to a tester strain.

We showed that, similar to results from yeast and mouse targeting experiments, targeting efficiency in Drosophila (1) was locus dependent; (2) could be improved by increasing donor:target homology; and (3) would be affected by sequence heterology between the donor and the target. We also showed some special features of Drosophila targeting: (1) targeting was much more efficient in the female germline than in the male germline, which might be related to the fact that Drosophila males do not have meiotic recombination; and (2) targeting frequencies were highly variable among different insertions of the donor P element, which might be caused by chromosomal position effects on donor generation. Using two different modifications of the original targeting scheme, we and others have succeeded in generating mutant alleles of different genes. We have also developed a two-step allelic replacement method that allowed us to recover a single target gene harboring the desired sequence modification starting with a tandem duplication of the target locus. We propose that the Drosophila targeting scheme should be adaptable to other organisms that have been successfully transformed. This is based on the facts that both the site-specific recombination system and the site-specific DSB system have been proven effective in many different heterologous systems.