Targeted gene replacement by homologous recombination in Drosophila stimulates production of second-site mutations

Gene replacement by homologous recombination is a powerful technique for generating mutations in Drosophila. While using this technique for the BEAF gene, we encountered non-targeted lethal mutations on the target chromosome that complicated the analysis of the BEAF mutations until they were discovered and removed by meiotic recombination. Subsequent experiments indicated that the gene-targeting method leads to a modest but significant three-fold increase in the rate of production of non-targeted lethal mutations. It is important to be aware of this phenomenon when using this method.