Multiplexed Transgenic Selection and Counterselection Strategies to Expedite Genetic Manipulation Workflows Using Drosophila melanogaster.

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 drugged fly food vials for transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 2: Determining the effective drug concentration for resistance and sensitivity markers used during transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 3: Preparing and confirming plasmid DNA for microinjection to perform transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 4: Microinjecting plasmid DNA into fly embryos to perform transgenic selection and counterselection strategies using D. melanogaster Basic Protocol 5: Crossing schemes to isolate desired progeny through transgenic selection and counterselection strategies using D. melanogaster.

Synthetic Assembly DNA Cloning to Build Plasmids for Multiplexed Transgenic Selection, Counterselection or Any Other Genetic Strategies Using Drosophila melanogaster.

We recently described a drug-based selectable and counterselectable genetic platform for the animal model system Drosophila melanogaster, consisting of four resistance and two sensitivity markers that allow direct selection for, or counterselection against, a desired genotype. This platform eliminates the need to identify modified progeny by traditional laborious screening using the dominant eye and body color markers, white+ and yellow+ , respectively. The four resistance markers permit selection of animals using G418 sulfate, puromycin HCl, blasticidin S, or hygromycin B, while the two sensitivity markers allow counterselection of animals against ganciclovir or acyclovir and 5-fluorocytosine. The six markers can be used alone or in combination to perform co-selection, combination selection, and counterselection, as well as co-counterselection. To make this novel selection and counterselection genetics platform easily accessible to and rapidly implementable by the scientific community, we used a synthetic assembly DNA cloning platform, GoldenBraid 2.0 (GB2.0). GB2.0 relies on two Type IIs restriction enzymes that are alternatingly used during successive cloning steps to make increasingly complex genetic constructs. Here we describe, as an example, how to perform synthetic assembly DNA cloning using GB2.0 to build such complex plasmids via the assembly of both components of the binary LexA/LexA-Op overexpression system, a G418 sulfate-selectable LexA transactivator plasmid, and a blasticidin S-selectable LexA-Op responder plasmid. We demonstrate the functionality of these plasmids by including the expression pattern obtained after co-injection, followed by co-selection using G418 sulfate and blasticidin S, resulting in co-transgenesis of both plasmids. Protocols are provided on how to obtain, adapt, and clone DNA parts for synthetic assembly cloning after de novo DNA synthesis or PCR amplification of desired DNA parts and how to assemble those DNA parts into multipartite transcription units, followed by how to further assemble multiple transcription units into genetic constructs of increasing complexity to perform multiplexed transgenic selection and counterselection, or any other genetic strategies using Drosophila melanogaster. The protocols we present can be easily adapted to incorporate any of the six selectable and counterselectable markers, or any other, markers, to generate plasmids of unmatched complexity for various genetic applications. A protocol on how to generate transgenic animals using these synthetically assembled plasmids is described in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Obtaining and cloning a de novo-synthesized DNA part for synthetic assembly DNA cloning Basic Protocol 2: Obtaining and cloning a DNA part amplified by PCR from existing DNA resources for synthetic assembly DNA cloning Alternate Protocol: Obtaining, adapting, and cloning a DNA part amplified by PCR from existing DNA resources for synthetic assembly DNA cloning Basic Protocol 3: Synthetic assembly DNA cloning of individual DNA parts into a multipartite transcription unit Basic Protocol 4: Synthetic assembly DNA cloning of multiple transcription units into genetic constructs of increasing complexity.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids.

Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.

Multiplex Hextuple Luciferase Assaying.

We recently expanded the commonly used dual luciferase assaying method toward multiplex hextuple luciferase assaying, allowing monitoring the activity of five experimental pathways against one control at the same time. In doing so, while our expanded assay utilizes a total of six orthogonal luciferases instead of two, this assay, conveniently, still utilizes the well-established reagents and principles of the widely used dual luciferase assay. Three quenchable D-luciferin-consuming luciferases are measured after addition of D-Luciferin substrate, followed by quenching of their bioluminescence (BL) and the measurement of three coelenterazine (CTZ)-consuming luciferases after addition of CTZ substrate, all in the same vessel. Here, we provide detailed protocols on how to perform such multiplex hextuple luciferase assaying to monitor cellular signal processing upstream of five transcription factors and their corresponding transcription factor-binding motifs, using a constitutive promoter as normalization control. The first protocol is provided on how to perform cell culture in preparation toward genetic or pharmaceutical perturbations, as well as transfecting a multiplex hextuple luciferase reporter vector encoding all luciferase reporter units needed for multiplex hextuple luciferase assaying. The second protocol details on how to execute multiplex hextuple luciferase assaying using a microplate reader appropriately equipped to detect the different BLs emitted by all six luciferases. Finally, the third protocol provides details on analyzing, plotting, and interpreting the data obtained by the microplate reader.

Determining effective drug concentrations for selection and counterselection genetics in Drosophila melanogaster.

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.

Multiplexed drug-based selection and counterselection genetic manipulations in Drosophila.

The 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.

A novel statistical method for interpreting the pathogenicity of rare variants.

PURPOSE: To achieve the ultimate goal of personalized treatment of patients, accurate molecular diagnosis and precise interpretation of the impact of genetic variants on gene function is essential. With sequencing cost becoming increasingly affordable, the accurate distinguishing of benign from pathogenic variants becomes the major bottleneck. Although large normal population sequence databases have become a key resource in filtering benign variants, they are not effective at filtering extremely rare variants. METHODS: To address this challenge, we developed a novel statistical test by combining sequencing data from a patient cohort with a normal control population database. By comparing the expected and observed allele frequency in the patient cohort, variants that are likely benign can be identified. RESULTS: The performance of this new method is evaluated on both simulated and real data sets coupled with experimental validation. As a result, we demonstrate this new test is well powered to identify benign variants, and is particularly effective for variants with low frequency in the normal population. CONCLUSION: Overall, as a general test that can be applied to any type of variants in the context of all Mendelian diseases, our work provides a general framework for filtering benign variants with very low population allele frequency.

Rapid and Efficient Synthetic Assembly of Multiplex Luciferase Reporter Plasmids for the Simultaneous Monitoring of Up to Six Cellular Signaling Pathways.

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 cell-based 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 cover large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, and their 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 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 stoichiometric ratios of each transcriptional unit in each transfected cell, resulting in lower experimental variation. Our proof-of-concept multiplex hextuple luciferase assay was designed to simultaneously monitor the p53, TGF-ß, NF-κß, c-Myc, and MAPK/JNK signaling pathways. The same synthetic assembly cloning pipeline allows the stitching of numerous other cellular pathway luciferase reporters. Here we present an improved three-step synthetic assembly protocol to quickly and efficiently generate multiplex hextuple luciferase reporter plasmids for other signaling pathways of interest. This improved assembly protocol provides the opportunity to analyze any five desired pathways at once much more quickly. Protocols are provided on how to prepare DNA components and destination vector plasmids, design synthetic DNA, perform assembly cloning of new transcriptional reporter elements, implement multipartite synthetic assembly cloning of single-pathway luciferase reporters, and carry out one-step assembly of final multiplex hextuple luciferase vectors. We present protocols on how to perform multiplex hextuple luciferase in an accompanying Current Protocols in Molecular Biology article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of DNA parts and destination vectors for synthetic assembly cloning Basic Protocol 2: DNA synthesis and assembly cloning of a typical transcriptional reporter element Alternate Protocol: DNA synthesis and assembly cloning of a challenging transcriptional reporter element Basic Protocol 3: Multipartite synthetic assembly cloning of individual pathway luciferase reporters Basic Protocol 4: One step assembly into final multiplex hextuple luciferase vectors Support Protocol: Generation of home-made chemocompetent E. coli DH10B-T1R cells.

Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying.

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 have expanded this approach to multiplex hextuple luciferase assays 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. Here, we present detailed protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κß, TGF-ß, p53, and MAPK/JNK) using the constitutive CMV promoter as normalization control. Protocols are provided for preparing reporter vector plasmids for multiplex reporter assaying, performing cell culture and multiplex luciferase reporter vector plasmid transfection, executing multiplex luciferase assays, and analyzing and interpreting data obtained by a plate reader appropriately equipped to detect the different luminescences. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of vectors for multiplex hextuple luciferase assaying Basic Protocol 2: Cell culture work for multiplex hextuple luciferase assays Basic Protocol 3: Transfection of luciferase reporter plasmids followed by drug and recombinant protein treatments Basic Protocol 4: Performing the multiplex hextuple luciferase assay.

A GoldenBraid cloning system for synthetic biology in social amoebae.

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.

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying.

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.