Current protocols最新文献

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 probe

Basic Protocol 2: Expression of the immunoproteasome in cells

Basic Protocol 3: Immunoproteasome probe application in live cells using a luminescent plate reader

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 screenings

Basic Protocol 2: Y1H screenings with arrayed gene libraries

Alternate Protocol: Automated screening with a liquid-handling robot

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: M^pro-On and -Off assay

Alternate Protocol 1: Virus production with transient P- and L TransIT transfection

Alternate Protocol 2: Virus production with transient P- and L Ca₂PO₄ transfection

Alternate Protocol 3: Luciferase-based variation of the On assay

Alternate Protocol 4: Screening assay with fluorescence-activated cell sorting readout

Support Protocol: Performing kinetic measurements with Off assay

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 spheroids

Basic Protocol 2: Organotypic spheroid fabrication

Basic Protocol 3: Vascularized spheroids on-chip

Basic Protocol 4: Functionality assays

Support Protocol 1: Cell Culture

Support Protocol 2: Immunocytochemistry

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 collection

Basic Protocol 2: Injection of stage-two one-cell medaka embryos

Basic 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

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 NPCs

Basic Protocol 2: Differentiation of NPCs into iAstrocytes

Support Protocol 1: Molecular validation of iAstrocytes

Support Protocol 2: Calcium imaging-based validation of iAstrocyte function

Support Protocol 3: Differentiation of NPCs into neurons

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 server

Basic Protocol 2: Predicting a structure conserved in two sequences with the RNAstructure web server

Alternative Protocol 1: Predicting a structure conserved in multiple sequences in the RNAstructure graphical user interface

Alternative Protocol 2: Predicting a structure conserved in two sequences with Dynalign in the RNAstructure graphical user interface

Alternative Protocol 3: Running TurboFold on the command line

Current Protocols is issuing corrections for the following protocol article.

Thomason, L. C., Costantino, N., Li, X., & Court, D. L. (2023). Recombineering: Genetic engineering in Escherichia coli using homologous recombination. Current Protocols, 3, e656. doi: 10.1002/cpz1.656

In the above-referenced article:

Figure 6 has been corrected so that the exclamation points now appear as degree symbols.

The current version online now includes these corrections and may be considered the authoritative version of record.

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) design

Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site

Basic Protocol 3: Cloning the sgRNA dual guide construct

Basic Protocol 4: Repair template design and cloning

Basic Protocol 5: Electroporation and selection of engineered B cells

Basic Protocol 6: Single-cell cloning of engineered B cells

Non-symbolic stimuli representing numerosities are invariably associated with continuous magnitudes, complicating the interpretation of experimental studies on numerosity perception. Although various algorithms for experimental numerosity generation have been proposed, they do not consider the quantifiable distribution of values of continuous magnitudes and the degree of numerosity-magnitudes association. Consequently, they cannot thoroughly exclude the possibility of magnitudes integration or strategy switch between different magnitudes in numerical stimulus perception. Here, we introduce a protocol for numerosity generation, animal training, and behavior outcomes analysis that takes the aforementioned issues into consideration. This protocol has been applied to rodents and is applicable to other animals in numerosity studies. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Algorithm for generating non-symbolic numerical stimuli

Alternate Protocol: General algorithm for generating non-symbolic numerical stimuli

Basic Protocol 2: Numerical training and testing for rodents