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 manipulations in vivo is a key strength of Drosophila, 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 of Drosophila genetics by allowing targeted combinatorial ectopic expression and knockdown of multiple genes from a single inducible transgene.
Most common human diseases are polygenic; they arise due to the disruption of multiple genes and are also influenced by environmental factors . For instance, genome landscapes of most solid tumors include concurrent somatic alterations in numerous oncogenes and tumor suppressors and many germline variants that can modify disease progression and response to therapy [2–4]. The ability to capture the genetic complexity and heterogeneity of human disease in experimental models is vital for the functional exploration of cancer omics datasets, improving our mechanistic understanding of disease biology and developing effective therapies. In addition to targeting multiple genes, in vivo disease modeling requires transgenes for tissue or cell-type specific and temporally regulated genetic manipulations and fluorescent labeling of targeted cells to study their interactions with their environment. Furthermore, parallel genetic perturbations within the local microenvironment or distant organs are often necessary to explore short-range and systemic interactions mediating disease phenotypes. While multiple such tools are available in genetically tractable model systems like Drosophila , bringing all these genetic tools together in experimental animals is challenging. It represents a significant barrier to in vivo disease modeling.
Materials and method
Cloning and Transgenesis
The 4[sh] clusters tester1-4 were synthesized by four rounds of PCRs using the primers listed in S1C Table. Guide and spacer sequences used in the construction are shown in S1A Table. Digitally assembled clusters and individual hairpins are shown in S1B Table. PCR products representing fully assembled [4sh] clusters were digested with XbaI (5’) and EcoRI (3’) introduced during the final round of PCR and cloned into the pWALIUM-Multiple Cloning Site (MCS) of pWALIUM 3xUAS attB, which is designed for shRNA expression (S1A and S1B Fig). To generate the longer test clusters 8[sh], 12[sh], and 16[sh], three additional 4[sh] clusters representing hairpins 5–8, 9–12, and 13–16 were digitally assembled (S1D Table) and generated by gene synthesis (Genewiz). The sequence-confirmed fragments were PCR-amplified and stitched to the 4[sh] cluster tester-1 by sequential PCR reactions. The resulting 8[sh], 12[sh], and 16[sh] clusters were digested with XbaI (5’) and NotI (3’) and cloned into the pWAL-MCS of pWALIUM 3xUAS attB (S1A and S1B Fig). Inverted constructs were generated by PCR-amplifying the 8[sh] and 16[sh] clusters from the multigenic vectors used for transgenesis using a new set of primers designed to append NotI and EcoRI restriction enzyme sites at the 5’ and the 3’ ends of the clusters respectively so that the clusters could be re-cloned into the MCS of the multigenic vector in the inverted orientation. The final products were sequence confirmed (Genewiz), and transgenic flies were generated by PhiC31-mediated targeted integration into the attp2 landing site on the third Drosophila chromosome  (BestGene).
Evaluating knockdown efficacy and positional effects within multi[sh] clusters
We have previously demonstrated that eight genes can be effectively knocked down from a single synthetic short-hairpin (sh) cluster . To determine whether knockdown efficacy is altered in the context of a multi[sh] cluster compared to single shRNA expression, we built a series of 4[sh] test clusters by stitching together short hairpin sequences available as transgenic UAS-shRNA fly lines from the TRiP collection . To this end, we selected three hairpin sequences targeting the Drosophila white (w), singed (sn), and the exogenous GFP gene, all of which provided strong knockdown of their target genes at the RNA, protein, or phenotypic level upon ubiquitous expression. We also included a short hairpin targeting the Drosophila p53 gene, which did not provide significant knockdown as a single hairpin, as a control (Fig 1 and S1A Table). To determine whether there are positional effects within a multi-hairpin cluster that might influence knockdown efficacy, we stitched together these four short hairpins to create four different 4[sh] clusters, UAS-tester1-4, where each short hairpin occupied a different position in the cluster (Fig 1A and S1B and S1C Table).
In recent years, in vivo combinatorial genetic perturbations have become increasingly essential to generate experimental models that capture the genetic complexity and heterogeneity of human diseases and for detailed mechanistic explorations of local and systemic interactions underlying disease states. Here, we report a new design that combines elements from different genetic manipulation tools for targeted, inducible expression and knockdown of multiple genes. This highly flexible and adaptable tool consolidates multiple genetic perturbations into a single, inducible polycistronic transgene to further increase the genetic sophistication of disease models. The ability to use a single UAS cassette for multiple genetic manipulations provides an opportunity to introduce additional transgenes, reporters, and lineage-tracing constructs into the multigenic vector. It also makes it broadly adaptable to other targeted expression, site-specific recombination, and gene editing platforms for mechanistic studies.
We thank Dr. Brian Washburn, Dr. Diego Zorio, and Cheryl Pye at the Florida State University Molecular Cloning Facility and Jason Cassara for technical support. This study used transgenic RNAi lines (Office of the Director R24 OD030002: "TRiP resources for modeling human disease") obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537).
Citation: Teague AG, Quintero M, Karimi Dermani F, Cagan RL, Bangi E (2023) A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila. PLoS Genet 19(6): e1010792. https://doi.org/10.1371/journal.pgen.1010792
Editor: Ken M. Cadigan, University of Michigan, UNITED STATES
Received: April 6, 2023; Accepted: May 18, 2023; Published: June 2, 2023
Copyright: © 2023 Teague et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by grants R03 CA219321 (EB) and R21 GM141734 (EB) from the National Institutes of Health. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.