Current protocols最新文献

Analysis of Free Oligosaccharides in Urine by High-Performance Liquid Chromatography–Tandem Mass Spectrometry 利用高效液相色谱-串联质谱法分析尿液中的游离低聚糖

Current protocols Pub Date : 2024-11-22 DOI: 10.1002/cpz1.70055

J. Daniel Sharer, Rongrong Huang, Timothy Wood, Paula Huffman, Yang Yan, Chelsea Zimmerman, Laura Pollard

引用次数: 0

Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome 免疫蛋白酶体笼式生物发光探针的合成与应用

Current protocols Pub Date : 2024-11-22 DOI: 10.1002/cpz1.70057

Cody A. Loy, Darci J. Trader

{"title":"Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome","authors":"Cody A. Loy, Darci J. Trader","doi":"10.1002/cpz1.70057","DOIUrl":"10.1002/cpz1.70057","url":null,"abstract":"Monitoring the catalytic activity of the proteasome and its various isoforms has become increasingly important with the continued development of core particle inhibitors and targeted protein degraders as potential therapies for diseases with high protein accumulation. The immunoproteasome (iCP) is expressed in a variety of diseases due to inflammatory signals, such as interferon-gamma, that alert the cell to begin generating iCP preferentially over the standard proteasome. There is a need to understand iCP activity and expression both in cells and in vivo because it is becoming a widely targeted isoform in a variety of diseases. Activity-based probes for the iCP have been developed, but their application has been limited due to their difficult synthesis and choice of fluorescent reporter. There has yet to be a selective iCP probe developed that incorporates a luminescent reporter that could be applied to a variety of different applications. The protocols presented here describe the synthesis of a cleavable activity-based bioluminescent probe that is selective for the iCP, and the application of the synthesized probe in immunoproteasome activity assays using a luminescent plate reader. Having this bioluminescent reporter, a better understanding of how the iCP is implicated in disease progression, as well as identification of small molecule interactors, can be achieved. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Synthesis of a bioluminescent immunoproteasome probeBasic Protocol 2: Expression of the immunoproteasome in cellsBasic Protocol 3: Immunoproteasome probe application in live cells using a luminescent plate reader","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142690025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast 在酵母中选择功能性启动子 DNA 诱饵和筛选阵列基因库的工作流程。

Current protocols Pub Date : 2024-11-21 DOI: 10.1002/cpz1.70059

Iris Fañanás-Pueyo, Ana-Mariya Anhel, Ángel Goñi-Moreno, Luis Oñate-Sánchez, Gerardo Carrera-Castaño

{"title":"Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast","authors":"Iris Fañanás-Pueyo, Ana-Mariya Anhel, Ángel Goñi-Moreno, Luis Oñate-Sánchez, Gerardo Carrera-Castaño","doi":"10.1002/cpz1.70059","DOIUrl":"10.1002/cpz1.70059","url":null,"abstract":"The yeast one-hybrid system (Y1H) is used extensively to identify DNA–protein interactions. The generation of large collections of open reading frames (ORFs) to be used as prey in screenings is not a bottleneck nowadays and can be carried out in-house or offered as a service by companies. However, the straightforward use of full gene promoters as baits to identify interacting proteins undermines the accuracy and sensitivity of the assay, especially in the case of multicellular eukaryotes. Therefore, it is paramount to implement procedures for efficient identification of suitable promoter fragments compatible with the Y1H assay. Here, we describe a workflow to identify biologically relevant conserved promoter fragments of Arabidopsis thaliana through simple and robust phylogenetic analyses. Additionally, we describe a manual method and its automated robotized version for rapid and efficient high-throughput Y1H screenings of arrayed ORF libraries with the identified DNA fragments. Moreover, this method can be scaled up or down and used for yeast two-hybrid screenings to search for possible interactors of proteins identified by the Y1H approach or any other protein of interest, altogether underscoring its suitability to build gene regulatory networks. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Selection of DNA baits for Y1H screeningsBasic Protocol 2: Y1H screenings with arrayed gene librariesAlternate Protocol: Automated screening with a liquid-handling robot","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Vesicular Stomatitis Virus as a Platform for Protease Activity Measurements 作为蛋白酶活性测量平台的水泡性口炎病毒

Current protocols Pub Date : 2024-11-21 DOI: 10.1002/cpz1.70062

Stefanie Rauch, Francesco Costacurta, Dorothee von Laer, Emmanuel Heilmann

{"title":"Vesicular Stomatitis Virus as a Platform for Protease Activity Measurements","authors":"Stefanie Rauch, Francesco Costacurta, Dorothee von Laer, Emmanuel Heilmann","doi":"10.1002/cpz1.70062","DOIUrl":"10.1002/cpz1.70062","url":null,"abstract":"Protease inhibitors are among the most powerful antiviral drugs. They have been used successfully against viruses, such as the human immunodeficiency virus (HIV), hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protease inhibitor screening tools are therefore important to identify inhibitors that have the potential to become antiviral drugs. In this article, we describe newly developed cell- and virus replicon-based platforms to screen inhibitors. We developed the methods presented here by genetically modifying vesicular stomatitis virus, a model virus from the family Rhabdoviridae. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Mpro-On and -Off assayAlternate Protocol 1: Virus production with transient P- and L TransIT transfectionAlternate Protocol 2: Virus production with transient P- and L Ca2PO4 transfectionAlternate Protocol 3: Luciferase-based variation of the On assayAlternate Protocol 4: Screening assay with fluorescence-activated cell sorting readoutSupport Protocol: Performing kinetic measurements with Off assay","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70062","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering and Evaluating Vascularized Organotypic Spheroids On-Chip 芯片上血管有组织球体的工程设计与评估

Current protocols Pub Date : 2024-11-21 DOI: 10.1002/cpz1.70058

James J. Tronolone, Nadin Mohamed, Christopher P. Chaftari, Yuxiang Sun, Tanmay Mathur, Abhishek Jain

{"title":"Engineering and Evaluating Vascularized Organotypic Spheroids On-Chip","authors":"James J. Tronolone, Nadin Mohamed, Christopher P. Chaftari, Yuxiang Sun, Tanmay Mathur, Abhishek Jain","doi":"10.1002/cpz1.70058","DOIUrl":"10.1002/cpz1.70058","url":null,"abstract":"Organotypic spheroids are evolving as a mainstream in vitro modeling platform, but it is crucial to integrate vascular tissue and perfusion for maintaining their longevity, stability, and physiological relevance. Current vascularization methods remain underdeveloped, and several protocols are poorly reproducible and are limited to use by a few select groups who have designed these methods. To achieve standardization, we offer a step-by-step guide to vascularize organotypic spheroids in case studies of pancreatic islets and cancer spheroids. Our systematic approach spans microfluidic chip design, spheroid fabrication, and vascularization techniques (vasculogenesis and angiogenesis) while describing critical tissue engineering methods. We also include additional insights and operating guidelines within our protocols that characterize and quantitate these models with molecular assays as well as our integrated computational algorithms of mass transport through formed capillary vessels. These protocols contribute to establishing reproducibility, standardization, and enhanced adoption by other contemporary organ-chip researchers, who want to engineer vascularized organoid-based microphysiological platforms. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Design and fabrication of microfluidic chips for vascularized spheroidsBasic Protocol 2: Organotypic spheroid fabricationBasic Protocol 3: Vascularized spheroids on-chipBasic Protocol 4: Functionality assaysSupport Protocol 1: Cell CultureSupport Protocol 2: Immunocytochemistry","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Injection of Ortho-Functionalized Tetrafluorinated Azobenzene-Containing siRNAs into Japanese Medaka Embryos for Photocontrolled Gene Silencing 向日本青鳉胚胎注射含 siRNA 的正功能化四氟偶氮苯，实现光控基因沉默。

Current protocols Pub Date : 2024-11-15 DOI: 10.1002/cpz1.70051

Makenzie Mateus, Matthew L. Hammill, Denina B. D. Simmons, Jean-Paul Desaulniers

{"title":"Injection of Ortho-Functionalized Tetrafluorinated Azobenzene-Containing siRNAs into Japanese Medaka Embryos for Photocontrolled Gene Silencing","authors":"Makenzie Mateus, Matthew L. Hammill, Denina B. D. Simmons, Jean-Paul Desaulniers","doi":"10.1002/cpz1.70051","DOIUrl":"10.1002/cpz1.70051","url":null,"abstract":"This article describes the detailed methodology of how to inject photoswitchable ortho-functionalized tetrafluorinated short interfering RNAs (F-siRNAs) into a single cell of stage-two Japanese medaka (Oryzias latipes) embryos and how to control gene silencing with different wavelengths of light. Many of the prior papers describing Japanese medaka embryo injections omit key information. As such, this article aims to give an in-depth explanation as to how the NanoJect III microinjector can be used for this purpose. To obtain the embryos for microinjection, adult medaka are housed under a 14-hr light, 10-hr dark cycle to mimic their natural breeding period. This induces mating at approximately the same time each day, when the lights turn on, so recently fertilized eggs can be obtained. Synthetic F-siRNAs are injected into transgenic stage-two single-cell Japanese medaka embryos expressing enhanced green fluorescent protein (eGFP). Our data demonstrate that our F-siRNAs can silence gene activity in Japanese medaka embryos expressing eGFP. Moreover, gene expression can be activated by exposing F-siRNA-injected embryos to blue light and deactivated a few days after exposure to green light. To the best of our knowledge, this marks the first reversible control of a gene-silencing oligonucleotide within an in vivo system. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Medaka maintenance and embryo collectionBasic Protocol 2: Injection of stage-two one-cell medaka embryosBasic Protocol 3: Evaluation of the F-siRNA gene-silencing ability through light activation and inactivation using blue and green light by measuring enhanced green fluorescent protein fluorescence","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70051","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generating iAstrocytes From Human Induced Pluripotent Stem Cells by Combining Low-Density Passaging of Neural Progenitor Cells and Transcription Factor NFIA Transdifferentiation 将神经祖细胞的低密度传代与转录因子 NFIA 的转分化相结合，从人类诱导多能干细胞中生成 iAstrocytes。

Current protocols Pub Date : 2024-11-15 DOI: 10.1002/cpz1.70049

Patrick Bosco, Ugur Akcan, Damian Williams, Heather M. Buchanan, Dritan Agalliu, Andrew A. Sproul

{"title":"Generating iAstrocytes From Human Induced Pluripotent Stem Cells by Combining Low-Density Passaging of Neural Progenitor Cells and Transcription Factor NFIA Transdifferentiation","authors":"Patrick Bosco, Ugur Akcan, Damian Williams, Heather M. Buchanan, Dritan Agalliu, Andrew A. Sproul","doi":"10.1002/cpz1.70049","DOIUrl":"10.1002/cpz1.70049","url":null,"abstract":"Astrocytes are key regulators of central nervous system (CNS) homeostasis, and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from human induced pluripotent stem cells (hiPSCs) using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediate allows for facile differentiation into both purified neuronal and astrocyte cell types in parallel from the same genetic background, depending on the experimental needs. This article presents a protocol to generate NPCs from hiPSCs, which are then differentiated into hiPSC-derived astrocytes, termed iAstrocytes. The resulting iAstrocytes express key markers of astrocyte identity at transcript and protein levels by bulk RNA-Seq and immunocytochemistry, respectively. Additionally, they respond to the inflammatory stimuli poly(I:C) and generate waves of calcium activity in response to either physical activity or the addition of ATP. Our approach offers a simple and robust method to generate and characterize human astrocytes, which can be used to model human disease affecting this cell type. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Differentiation of hiPSCs to NPCsBasic Protocol 2: Differentiation of NPCs into iAstrocytesSupport Protocol 1: Molecular validation of iAstrocytesSupport Protocol 2: Calcium imaging-based validation of iAstrocyte functionSupport Protocol 3: Differentiation of NPCs into neurons","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142638765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Using the RNAstructure Software Package to Predict Conserved RNA Structures 使用 RNAstructure 软件包预测保守的 RNA 结构。

Current protocols Pub Date : 2024-11-14 DOI: 10.1002/cpz1.70054

Abhinav Mittal, Sara E. Ali, David H. Mathews

{"title":"Using the RNAstructure Software Package to Predict Conserved RNA Structures","authors":"Abhinav Mittal, Sara E. Ali, David H. Mathews","doi":"10.1002/cpz1.70054","DOIUrl":"10.1002/cpz1.70054","url":null,"abstract":"The structures of many non-coding RNAs (ncRNA) are conserved by evolution to a greater extent than their sequences. By predicting the conserved structure of two or more homologous sequences, the accuracy of secondary structure prediction can be improved as compared to structure prediction for a single sequence. Here, we provide protocols for the use of four programs in the RNAstructure suite to predict conserved structures: Multilign, TurboFold, Dynalign, and PARTS. TurboFold iteratively aligns multiple homologous sequences and estimates the pairing probabilities for the conserved structure. Dynalign, PARTS, and Multilign are dynamic programming algorithms that simultaneously align sequences and identify the common secondary structure. Dynalign uses a pair of homologs and finds the lowest free energy common structure. PARTS uses a pair of homologs and estimates pairing probabilities from the base pairing probabilities estimated for each sequence. Multilign uses two or more homologs and finds the lowest free energy common structure using multiple pairwise calculations with Dynalign. It scales linearly with the number of sequences. We outline the strengths of each program. These programs can be run through web servers, on the command line, or with graphical user interfaces. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Predicting a structure conserved in three or more sequences with the RNAstructure web serverBasic Protocol 2: Predicting a structure conserved in two sequences with the RNAstructure web serverAlternative Protocol 1: Predicting a structure conserved in multiple sequences in the RNAstructure graphical user interfaceAlternative Protocol 2: Predicting a structure conserved in two sequences with Dynalign in the RNAstructure graphical user interfaceAlternative Protocol 3: Running TurboFold on the command line","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634986","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Correction: Recombineering: Genetic Engineering in Escherichia coli Using Homologous Recombination 更正：重组：利用同源重组在大肠杆菌中进行基因工程。

Current protocols Pub Date : 2024-11-14 DOI: 10.1002/cpz1.70064

Lynn C. Thomason, Nina Costantino, Xintian Li, Donald L. Court

引用次数: 0

Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells 操纵人类 B 淋巴瘤细胞内源基因的高效 CRISPR/Cas9 基因敲入方法。

Current protocols Pub Date : 2024-11-13 DOI: 10.1002/cpz1.70041

Laura A. Murray-Nerger, Benjamin E. Gewurz

{"title":"Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells","authors":"Laura A. Murray-Nerger, Benjamin E. Gewurz","doi":"10.1002/cpz1.70041","DOIUrl":"10.1002/cpz1.70041","url":null,"abstract":"Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) designBasic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in siteBasic Protocol 3: Cloning the sgRNA dual guide constructBasic Protocol 4: Repair template design and cloningBasic Protocol 5: Electroporation and selection of engineered B cellsBasic Protocol 6: Single-cell cloning of engineered B cells","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0