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ABSTRACT
In this report, we describe several approaches to improve the scalability and throughput of major genetic crosses in ends-out gene targeting. We generated new sets of targeting vectors and fly stocks and introduced a novel negative selection marker that drastically reduced the frequency of false-positive targeting candidates.
THE development of homologous recombinationbased gene targeting is a landmark breakthrough in Drosophila genetics (Rong and Golic 2000; Gong and Golic 2003). In particular, the so-called "ends-out" or replacement-type gene targeting offers a straightforward approach for generating either knockout or knockin alleles. To date, there are already .20 genes that have been modified by ends-out targeting (supplemental Table 1). Nonetheless, the frequency of targetspecific homologous recombination in Drosophila varies tremendously, ranging from .1/200 gametes (Manoli et al. 2005) to ,1/350,000 (Jones et al. 2007) (also Y. Hong, unpublished data), i.e., a .1800-fold difference. In cases of low targeting efficiency (,1/100,000 gametes), ends-out targeting can be exceedingly time and labor intensive. Here,we optimized the current ends-out targeting scheme by focusing on improving the scalability andthroughput of its major genetic crosses. As illustrated in Figure 1a, there are three major genetic crosses in a typical ends-out targeting. In the targeting cross, virgin females of a transgenic line bearing the donorDNA("P{donor}") are crossed with hs-FLP, hs-I-SceI males, and their larval progeny are heat-shocked to induce the generation of linear donorDNAfragments by FLPase and I-SceI enzymes. In the screening cross, virgin females from the targeting cross that are of the correct genotype (P{donor}*/hs-FLP, hs-I-SceI) are crossed with proper chromosome balancer males, and preliminary targeting candidates are recovered on the basis of their w1 marker. However, many of these candidatesmight be false positives due to the failure of excision or nontargeting integration of the donor DNA. In the mapping cross, only preliminary candidates whose w1 marker is mapped to the target gene chromosome are selected for further analysis.
For the targeting cross, the number of P{donor}*/hs-FLP, hs-I-SceI virgin females directly determines the scale of the whole targeting experiment. Genes that are resistant to homologous recombination may require collecting and sorting .15,000 virgins from the targeting cross (Larsson et al. 2004), which is extremely labor intensive due to the time-sensitive nature of virgin collection and the genotyping process. To eliminate this major...