A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in < i > Drosophila < /i >

by Alexander G. Teague, Maria Quintero, Fateme Karimi Dermani, Ross L. Cagan, Erdem Bangi Experimental models that capture the genetic complexity of human disease and allow mechanistic explorations of the underlying cell, tissue, and organ interactions are crucial to furthering our understanding of disease biology. Such models require combinatorial manipulations of multiple genes, often in more than one tissue at once. The ability to perform complex genetic manipulationsin vivo is a key strength ofDrosophila, where many tools for sophisticated and orthogonal genetic perturbations exist. However, combining the large number of transgenes required to establish more representative disease models and conducting mechanistic studies in these already complex genetic backgrounds is challenging. Here we present a design that pushes the limits ofDrosophila genetics by allowing targeted combinatorial ectopic expression and knockdown of multiple genes from a single inducible transgene. The polycistronic transcript encoded by this transgene includes a synthetic short hairpin cluster cloned within an intron placed at the 5 ’ end of the transcript, followed by two protein-coding sequences separated by the T2A sequence that mediates ribosome skipping. This technology is particularly useful for modeling genetically complex diseases like cancer, which typically involve concurrent activation of multiple oncogenes and los s of multiple tumor suppressors. Furthermore, consolidating multiple gen...
Source: PLoS Genetics - Category: Genetics & Stem Cells Authors: Source Type: research