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Figure 7


Fig. 6. Gene targeting in Drosophila. (A) Ends-in insertional gene targeting. The donor construct, within a P element, contains a region of homology (the targeting construct, red) interrupted by a restriction recognition site for the meganuclease I-SceI and flanked by FRT recognition sites for FLP recombinase. It also contains a marker (white+) and an appropriately located restriction recognition site for the meganuclease I-CreI for a second round of homologous recombination. After P element transgenesis, a linearized episome is generated in vivo by FLP and I-SceI. Correct targeting results in white+ expression and a tandem duplication of the locus. This duplication can be reduced to single copy using I-CreI, resulting in loss of white+. (B) Ends-out replacement gene targeting. The donor construct, within a P element, contains a region of homology interrupted by a white+ marker and is flanked by restriction recognition sites for the meganuclease I-SceI and FRT recognition sites for FLP recombinase. After P element transgenesis, identified by white+, linearized targeting DNA is generated in vivo by FLP and I-SceI. Correct targeting results in a white+ phenotype and replacement of part of the locus.





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