Patent ID: 7285699

Claim:
A method of gene targeting in Drosophila comprising: (a) selecting a Drosophila target gene; (b) providing a donor construct comprising a transposon and a DNA segment homologous to the target gene comprising a marker sequence, wherein the DNA segment is flanked at both ends by a rare-cutting endonuclease recognition site and additionally flanked by a recombinase recognition site; (c) introducing the donor construct to a female Drosophila to obtain a transgenic female Drosophila whose genome comprises the transposon and the DNA segment flanked by said rare-cutting endonuclease recognition sites and said recombinase recognition sites; (d) providing a construct comprising a DNA sequence encoding a rare-cutting endonuclease and a DNA sequence encoding a recombinase, wherein each DNA sequence is operably linked to an inducible promoter; (e) introducing the construct of step (d) to a male Drosophila to obtain a transgenic male Drosophila whose genome comprises a DNA sequence encoding the rare-cutting endonuclease and a DNA sequence encoding the recombinase, wherein each DNA sequence is operably linked to an inducible promoter; (f) crossing the male transgenic Drosophila and the female transgenic Drosophila to produce progeny whose genome comprises the transposon and the DNA segment flanked by the rare-cutting endonuclease recognition sites and recombinase recognition sites, and whose genome also comprises a DNA sequence encoding the rare-cutting endonuclease and a DNA sequence encoding the recombinase, wherein each DNA sequence is operably linked to an inducible promoter; (g) activating the inducible promoter in the progeny of step (f) to express the rare-cutting endonuclease and the recombinase such that recombination occurs between the target gene and the target construct; and (h) selecting a transgenic progeny whose genome comprises the disrupted target gene.