A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila

Abstract

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. 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 loss of multiple tumor suppressors. Furthermore, consolidating multiple genetic perturbations into a single transgene further streamlines the ability to perform combinatorial genetic manipulations and makes it readily adaptable to a broad palette of transgenic systems. This flexible design for combinatorial genetic perturbations will also be a valuable tool for functionally exploring multigenic gene signatures identified from omics studies of human disease and creating humanized Drosophila models to characterize disease-associated variants in human genes. It can also be adapted for studying biological processes underlying normal tissue homeostasis and development that require simultaneous manipulation of many genes.

Fig 1. Evaluating knockdown efficacy and positional effects in the context of synthetic multi-hairpin clusters.

A. 4[sh] tester cluster design. The position of each short hairpin is varied so that all four short hairpins occupy all four possible positions within the cluster. B. Evaluation of sn and w knockdown efficacy using bristle and eye phenotypes observed in response to the ubiquitous expression of tester clusters and single hairpin controls. All phenotypes were fully penetrant (n = 50 animals). tub-gal4-only animals were used as baseline controls to demonstrate the wild-type eye and bristle phenotypes. C,D. Evaluation of p53 (C) and GFP (D) knockdown efficacy in response to cluster and single hairpin expression by western blotting. Error bars represent the standard error of the mean (SEM). *:p ≤0.05, **:p≤ 0.01, ***: p≤ 0.001, ****: p≤ 0.0001 (ordinary one-way ANOVA with Tukey’s multiple comparisons). Differences between the four tester constructs are not statistically significant.

Fig 2. Evaluating knockdown efficacy in the context of longer clusters.

A. Longer short hairpin cluster designs. 8[sh], 12[sh], 16[sh] clusters were generated by adding additional hairpins to the 4[sh] tester-1 cluster. Exogenous genes targeted by the clusters are in orange. B-G. Knockdown efficacy of short hairpins at indicated positions within longer clusters by qPCR (B,C,E-G) and western blot (D) analysis. Clusters that do not include the hairpin being evaluated are used as negative controls (4[sh] for D and E, 8[sh] for F, 12[sh] for G). Error bars represent the standard error of the mean (SEM). *:p ≤0.05, **:p≤ 0.01, ***: p≤ 0.001, ****: p≤ 0.0001 (ordinary one-way ANOVA with Tukey’s multiple comparisons).

Fig 3. Multi[sh] cluster design as a streamlined screening strategy to identify effective short hairpins for multiple tumor suppressors.

A. Four different 4[sh] clusters targeting Drosophila orthologs of four recurrently mutated colorectal cancer tumor suppressors TP53 (p53), APC (apc), SMAD4 (Med) and SMAD2 (smox). Each cluster expresses a different short hairpin targeting the same set of four genes. B. Evaluation of knockdown efficacy of p53, apc, Med and smox in response to the ubiquitous expression of the 4[sh] pams clusters by qPCR. C. An 8[sh] cluster generated by stitching pams-2 and pams-3 4[sh] clusters. D. Evaluation of knockdown efficacy of p53, apc, Med and smox in response to the ubiquitous expression of the 8[sh] pams cluster compared to pams-2 and pams-3 by qPCR. E. A 16[sh] cluster which includes 4 different hairpin sequences targeting each gene. F. Evaluation of knockdown efficacy of p53, apc, Med and smox in response to the ubiquitous expression of the 16[sh] pams cluster by qPCR. B,D,F. Error bars represent the standard error of the mean (SEM). *:p ≤0.05, **:p≤ 0.01, ***: p≤ 0.001, ****: p≤ 0.0001 (ordinary one-way ANOVA with Tukey’s multiple comparisons).

Fig 4. A polycistronic design for simultaneous gene knockdown and expression.

A. The construct designed for intron-mediated expression of the 4[sh] tester 1 cluster. B. Evaluation of sn and GFP knockdown in response to intron-mediated cluster expression. C. The bicistronic construct designed for T2A-mediated expression of Drosophila orthologs of two oncogenes commonly altered in human colon tumors, an oncogenic KRAS[G12V] (dRAS^G12V) and IRS2 (chico). The control construct where each coding sequence is cloned downstream of its own UAS enhancer/promoter in the multigenic vector. D. Evaluation of dRAS^G12V and Chico protein expression from the bicistronic construct by western blot analysis. E. The polycistronic construct designed for knockdowns of the 4 colorectal cancer tumor suppressors by intron-mediated expression of a 4[sh] cluster and T2A-mediated expression of two proteins from a single transcript. F,G. Evaluation of knockdown efficacy of p53, apc, Med and smox by qPCR (F) and dRAS^G12V and Chico protein expression (G) upon ubiquitous expression of the 6-hit polycistronic construct (E). B,F. Error bars represent the standard error of the mean (SEM). **:p≤ 0.01, ***: p≤ 0.001, ****: p≤ 0.0001 (ordinary one-way ANOVA with Tukey’s multiple comparisons).