DRUSE

Juan Huang and colleagues describe several approaches to improve crosses in ends-out gene targeting. They generated new sets of targeting vectors and fly stocks and introduced a novel negative selection marker, reducing the frequency of false-positive targeting candidates.

Figure 1. Genetic crosses in targeting experiments. (a) Genetic crosses of a typical ends-out targeting experiment. The transgenic donor DNA (“P{donor}“) is on the second chromosome, while the target gene is on the third chromosome. P{donor}*, linearized extrachromosomal donor DNA fragment that only exists transiently—it will either be lost permanently or inserted into a chromosome by targeted or nontargeted integration events. Target*, potential targeting events. Note that the majority of potential targeting events may be nonspecific and not located on the third chromosome (see text). ?, this copy of the second chromosome is inherited from the female in the screening cross. It could be the donor chromosome or hs-FLP, hs-I-SceI, or the recombinant between the two. Nonetheless, this copy of the chromosome is irrelevant in the mapping cross. (b) Genotypes of 6934-hid and 6935-hid. (c) 6934-hid stock eliminates the virgin collection and genotype sorting in the targeting cross. (d) The genetic cross scheme of the dArf6^KO targeting experiment. Because dArf6 is on the second chromosome, a transgenic line carrying dArf6^KO donor DNA (“P{dArf6}^Rpr+“) on the third chromosome was used. w; Pin/CyO; Gal4^221[w–] stock was used to set up the screening cross in lieu of a regular Pin/CyO balancer stock. This allowed simultaneous selection against nonspecific targeting candidates while balancing the potential specific targeting candidates from the screening cross. P{dArf6}^Rpr+*, linearized extrachromosomal dArf6^KO donor DNA fragment. dArf6^KO*, potential targeting events.

Huang J, Zhou W, Watson AM, Jan YN, Hong Y.
Efficient ends-out gene targeting in Drosophila.
Genetics. 2008 Sep;180(1):703-7. Epub 2008 Aug 30.