Case studies of ends-out gene targeting in Drosophila.

Ends-in and ends-out gene replacement approaches have been successfully used to disrupt Drosophila genes involved in a variety of biological processes. These methods combine double-strand breaks and homologous recombination to replace a targeted chromosome region with a designed DNA sequence. Unfortunately, these methods require large numbers of single animal crosses, making them both time consuming and labor intensive. Here, we designed a single complete targeting vector for use in a mass crossing ends-out gene targeting study. Importantly, our gene targeting method included a balancer chromosome to block endogenous homologous chromosome pairing and to promote pairing between the foreign targeting DNA fragment and the targeted chromosome. This technique provided successful and efficient gene replacement, greatly facilitating the gene knockout procedure. 2009 Wiley-Liss, Inc.