RESUMO
Neural stem cells, the source of newborn neurons in the adult hippocampus, are intimately involved in learning and memory, mood, and stress response. Despite considerable progress in understanding the biology of neural stem cells and neurogenesis, regulating the neural stem cell population precisely has remained elusive because we have lacked the specific targets to stimulate their proliferation and neurogenesis. The orphan nuclear receptor TLX/NR2E1 governs neural stem and progenitor cell self-renewal and proliferation, but the precise mechanism by which it accomplishes this is not well understood because its endogenous ligand is not known. Here, we identify oleic acid (18:1ω9 monounsaturated fatty acid) as such a ligand. We first show that oleic acid is critical for neural stem cell survival. Next, we demonstrate that it binds to TLX to convert it from a transcriptional repressor to a transcriptional activator of cell-cycle and neurogenesis genes, which in turn increases neural stem cell mitotic activity and drives hippocampal neurogenesis in mice. Interestingly, oleic acid-activated TLX strongly up-regulates cell cycle genes while only modestly up-regulating neurogenic genes. We propose a model in which sufficient quantities of this endogenous ligand must bind to TLX to trigger the switch to proliferation and drive the progeny toward neuronal lineage. Oleic acid thus serves as a metabolic regulator of TLX activity that can be used to selectively target neural stem cells, paving the way for future therapeutic manipulations to counteract pathogenic impairments of neurogenesis.
Assuntos
Hipocampo , Neurogênese , Ácido Oleico , Receptores Citoplasmáticos e Nucleares , Animais , Proliferação de Células , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Ligantes , Camundongos , Neurogênese/fisiologia , Ácido Oleico/metabolismo , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
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.
Assuntos
Drosophila melanogaster , Técnicas de Transferência de Genes , Animais , Drosophila melanogaster/genética , Animais Geneticamente Modificados , DNA , Drosophila/genética , Genômica , Clonagem Molecular , Células ClonaisRESUMO
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.
Assuntos
Drosophila melanogaster , Proteômica , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/genética , Fluxo de Trabalho , DNA , Drosophila/genéticaRESUMO
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.
Assuntos
DNA , Drosophila melanogaster , Animais , Drosophila melanogaster/genética , Clonagem Molecular , Animais Geneticamente Modificados/genética , Plasmídeos/genéticaRESUMO
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.
Assuntos
Drosophila melanogaster , Resistência a Medicamentos/genética , Engenharia Genética/métodos , Animais , Animais Geneticamente Modificados/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Feminino , Marcadores Genéticos/genética , MasculinoRESUMO
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.