Comparing selection schemes in BAC - recombineering method of tagging a novel Drosophila gene

Sialylation plays an important role in the Drosophila nervous system. However, the regulation of sialylation pathway remains poorly understood. We focused our analysis on a novel gene, CMP-Sialic acid synthetase (CSAS), that is predicted to be a key player of the pathway. In order to investigate its expression, we decided to introduce a tag sequence into the CSAS genomic locus within a BAC clone using recombineering strategy. We wanted to introduce the tag without any additional extraneous sequences in order to minimize the influence of the insert on the gene’s function. We sought to modify existing recombineering protocols and test different selection and screening methods during recombineering. Our results confirmed the general utility of positive/negative selection approach using rpsL-kana marker. However, they also revealed the limitation of this strategy, as it did not allow unambiguously to identify recombinant clones, while resulting in enrichment rather than selection for desired recombineering events. Research Notes Dros. Inf. Serv. 94 (2011) 116