Multiplexed drug-based selection and counterselection genetic manipulations in Drosophila

The power of Drosophila melanogaster as a model system relies on tractable germline genetic manipulations. Despite Drosophila’s expansive genetics toolbox, such manipulations are still accomplished one change at a time and depend predominantly on phenotypic screening. We describe a drug-based genetic platform consisting of four selection and two counterselection markers, eliminating the need to screen for modified progeny. These markers work reliably individually or in combination to produce specific genetic outcomes. We demonstrate three example applications of multiplexed drug-based genetics by generating (1) transgenic animals, expressing both components of binary overexpression systems in a single transgenesis step; (2) dual selectable and counterselectable balancer chromosomes; and (3) selectable, fluorescently tagged P[acman] bacterial artificial chromosome (BAC) strains. We perform immunoprecipitation followed by proteomic analysis on one tagged BAC line, demonstrating our platform’s applicability to biological discovery. Lastly, we provide a plasmid library resource to facilitate custom transgene design and technology transfer to other model systems. [Display omitted] •In vivo analysis of four selection and two counterselection markers•Individual or multiplexed use of markers to produce complex genotypes•Demonstrating usefulness of markers through three separate applications•Provision of DNA and vector resources to facilitate future applications Matinyan et al. describe a set of four drug selection and two drug counterselection markers for use in Drosophila genetics. The orthogonal markers can be used individually or combined for complex, multiplex genetic manipulations. Use of drug selection and counterselection eliminates screening efforts for modified animals, saving time and cost.