Abstract
SUMMARYThe 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.
Highlights
Genetic engineering technologies in Drosophila melanogaster have greatly advanced the study of basic biology and human disease (Bier, 2005; Venken and Bellen, 2007, 2014; Venken et al, 2016; Bellen et al, 2019; Link and Bellen, 2020)
This plasmid carrying a transgene coupled to a dominant physical marker is injected into early-stage, syncytial embryos targeting the future germline in a genetic background deficient for the marker (Venken and Bellen, 2007)
We describe a drug-based selection and counterselection platform for multiplexed genetic manipulations in Drosophila melanogaster that is readily transferrable to other model systems
Summary
Genetic engineering technologies in Drosophila melanogaster have greatly advanced the study of basic biology and human disease (Bier, 2005; Venken and Bellen, 2007, 2014; Venken et al, 2016; Bellen et al, 2019; Link and Bellen, 2020). Germline genetic manipulations in fruit flies, e.g., insertional mutagenesis and transgenesis, almost exclusively relied on P element transposons (Ryder and Russell, 2003). They were either remobilized from one location in the genome to another (Cooley et al, 1988) or jumped into the genome from a microinjected plasmid (Rubin and Spradling, 1982). Introducing tractable genetic modifications into flies starts with microinjecting a plasmid (Venken and Bellen, 2007). Screening genetically modified progeny can be time consuming and laborious (Venken et al, 2006, 2009, 2010; Venken and Bellen, 2007; Dahmann, 2008; Beumer and Carroll, 2014; Bier et al, 2018)
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