Highlights d In vivo analysis of four selection and two counterselection markers d Individual or multiplexed use of markers to produce complex genotypes d Demonstrating usefulness of markers through three separate applications

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.

Summary We recently integrated into fly genetics a set of four selection and two counterselection markers and their corresponding drugs that can be used individually or in combination. These markers eliminate the need to visually screen progeny. Before using these markers in new genetic backgrounds, effective selection/counterselection concentrations should be established for each marker/drug combination. This protocol describes how to set up, perform, and analyze a drug titration curve to determine the effective selection/counterselection drug concentrations for their corresponding markers. For complete details on the use and execution of this protocol, please refer to Matinyan et al., 2021 .

We recently described a set of four selectable and two counterselectable markers that provide resistance and sensitivity, respectively, against their corresponding drugs using the model organism Drosophila melanogaster. The four selectable markers provide animals with resistance against G418 sulfate, puromycin HCl, blasticidin S, or hygromycin B, whereas the two counterselection markers make animals sensitive to ganciclovir/acyclovir or 5‐fluorocytosine. Unlike classical phenotypic markers, whether visual or fluorescent, which require extensive screening of progeny of a genetic cross for desired genotypes, resistance and sensitivity markers eliminate this laborious procedure by directly selecting for, or counterselecting against, the desired genotypes. We demonstrated the usefulness of these markers with three applications: 1) generating dual transgenic animals for binary overexpression (e.g., GAL4/UAS) analysis in a single step through the process of co‐injection, followed by co‐selection resulting in co‐transgenesis; 2) obtaining balancer chromosomes that are both selectable and counterselectable to manipulate crossing schemes for, or against, the presence of the modified balancer chromosome; and 3) making both selectable and fluorescently tagged P[acman] BAC transgenic animals for gene expression and proteomic analysis. Here, we describe detailed procedures for how to use these drug‐based selection and counterselection markers in the fruit fly D. melanogaster when making dual transgenic animals for binary overexpression as an example. Dual transgenesis integrates site‐specifically into two sites in the genome in a single step, namely both components of the binary GAL4/UAS overexpression system, via a G418 sulfate–selectable GAL4 transactivator plasmid and a blasticidin S–selectable UAS responder plasmid. The process involves co‐injecting the two plasmids, followed by co‐selection using G418 sulfate and blasticidin S, resulting in co‐transgenesis of the two plasmids in the fly genome. We demonstrate the functionality of the procedure based on the expression pattern obtained after dual transgenesis of the two plasmids. We provide protocols on how to prepare drugged fly food vials, determine the effective drug concentration for markers used during transgenic selection and counterselection strategies, and prepare and confirm plasmid DNA for microinjection, followed by the microinjection procedure itself and setting up crossing schemes to isolate desired progeny through selection and/or counterselection. These protocols can be easily adapted to any combination of the six selectable and counterselectable markers we described or any new marker that is resistant or sensitive to a novel drug. Protocols on how to build plasmids by synthetic‐assembly DNA cloning or modify plasmids by serial recombineering to perform a plethora of selection, counterselection, or any other genetic strategies are presented in two accompanying Current Protocols articles. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing drug...

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases have arisen as possible genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. Here, we expanded luciferase multiplexing in post-lysis endpoint luciferase assays from two towards six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. Using synthetic assembly cloning, all six luciferase reporter units are stitched together into one plasmid; facilitating delivery of all reporter units through a process we named solotransfection, minimizing experimental errors. We engineered a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We were able to monitor the effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways.We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, as well as its broad application across different research fields.Running title: Multiplex hextuple luciferase assaying -3-

GoldenBraid is a rapid, modular, and robust cloning system used to assemble and combine genetic elements. Dictyostelium amoebae represent an intriguing synthetic biological chassis with tractable applications in development, chemotaxis, bacteria–host interactions, and allorecognition. We present GoldenBraid as a synthetic biological framework for Dictyostelium, including a library of 250 DNA parts and assemblies and a proof-of-concept strain that illustrates cAMP-chemotaxis with four fluorescent reporters coded by one plasmid.

High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. While most cellbased assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. The signals from such measurements should be independently detectable and measure large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, whose output signals can be sensitively detected. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously. We used synthetic assembly cloning (Vazquez-Vilar, et al., 2020) [*Copy Editor: Please ask Au to confirm that this should be cited here; he removed mention of "GoldenBraid" in this sentence during revision, so just double-checking.] to assemble all six luciferase reporter units into a single vector, over eight stitching rounds. Because all six reporters are on a single piece of DNA, a single vector ensures

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway, and a second that is coupled to a control pathway for normalization purposes. We expanded this approach to multiplex hextuple luciferase assaying that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters.