Current protocols最新文献

{"title":"Suboccipital Cisterna Magna Injection for Vehicle Delivery in Pigs Using Computed Tomography","authors":"Luke S. Myers,&nbsp;Sarah Christian,&nbsp;Jennifer Fridley,&nbsp;Scott V. Dindot","doi":"10.1002/cpz1.70069","DOIUrl":"10.1002/cpz1.70069","url":null,"abstract":"<p>Gene therapies are being developed for several central nervous system (CNS) disorders. These therapies are primarily administered to the CNS via the cerebrospinal fluid (CSF), as the blood–brain barrier prevents the transport of large molecules to the brain. Currently, intrathecal injection is the most commonly used route of administration over cisterna magna injections in the clinic for gaining access to the CSF. However, studies in nonhuman primates (NHPs) have shown that administering gene therapies via suboccipital cisterna magna injection results in superior distribution and more cells being transduced in the brain compared to lumbar injection. It has also been reported that comparable CNS size is important when translating therapeutic dosages from animal studies to human trials. Therefore, we chose to develop a computed tomography (CT)-guided cisterna magna injection protocol in pigs as they are anatomically closer in size to humans than nonhuman primates and rodents. Pigs are also a readily available and cost-effective large animal model for preclinical studies compared to nonhuman NHPs. In this paper, we describe a method for CT-guided suboccipital cisterna magna injections in pigs. We developed this protocol utilizing CT to confirm needle placement with three-dimensional visualization. A CT-guided injection minimizes procedural risk and can be performed without a contrast agent, which is required in magnetic resonance and fluoroscopy imaging. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Computed tomography–guided suboccipital cisterna magna injection in pigs to confirm needle placement prior to the administration of a test article or vehicle</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142803659","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}

{"title":"Isolation and Molecular Profiling of Nuclei of Specific Neuronal Types from Human Cerebral Cortex and Striatum","authors":"Christina Pressl,&nbsp;Matthew Baffuto,&nbsp;Paul Darnell,&nbsp;Cuidong Wang,&nbsp;Thomas S. Carroll,&nbsp;Nathaniel Heintz,&nbsp;Kert Mätlik","doi":"10.1002/cpz1.70067","DOIUrl":"10.1002/cpz1.70067","url":null,"abstract":"<p>Most pathological conditions of the central nervous system do not affect all cell types to the same extent. Delineation of molecular events underlying disease symptoms, including genetic, epigenetic, and transcriptional changes, thus relies on the ability to characterize a specific cell type separately from others. We have developed a methodology for the collection of nuclear RNA and genomic DNA of specific cell types from frozen post-mortem striatum and cerebral cortex. This allows deep transcriptomic profiling of specific cell populations and characterization of their genomes and epigenetic properties. The method is based on the purification of cell nuclei, followed by fluorescence-activated sorting of nuclei (FANS) labeled with nucleic acid probes or antibodies binding to targets present in specific cell types. The protocol describes in detail the procedure for isolating and labeling neuronal and glial nuclei from human brain tissue, the steps that can be taken to extract RNA and genomic DNA, a way to combine the procedure with ATAC-seq to yield information about chromatin accessibility, as well as computational measures for assessing the quality of cell type-specific RNA-seq and ATAC-seq datasets. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Tissue homogenization, isolation of cell nuclei by ultracentrifugation and formaldehyde-fixation</p><p><b>Basic Protocol 2</b>: Antibody-based labeling and isolation of nuclei of specific neocortical neuron types</p><p><b>Support Protocol 1</b>: Generation of ATAC-seq libraries from the nuclei of specific neuron types of the cerebral cortex</p><p><b>Basic Protocol 3</b>: Nucleic acid hybridization-based labeling and isolation of nuclei of specific striatal projection neuron types</p><p><b>Alternate Protocol 1</b>: Labeling and isolation of nuclei of specific striatal interneuron types</p><p><b>Support Protocol 2</b>: Generation of ATAC-seq libraries from the nuclei of specific striatal neuron types</p><p><b>Basic Protocol 4</b>: Extraction of genomic DNA and nuclear RNA and preparation of sequencing libraries</p><p><b>Basic Protocol 5</b>: Processing and quality control of FANS-seq and ATAC-seq data</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11627125/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142803658","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}

{"title":"Visualizing Volumetric and Segmentation Data using Mol* Volumes & Segmentations 2.0","authors":"Aliaksei Chareshneu,&nbsp;Alessio Cantara,&nbsp;Dominick Tichý,&nbsp;David Sehnal","doi":"10.1002/cpz1.70070","DOIUrl":"10.1002/cpz1.70070","url":null,"abstract":"<p>Ever-increasing availability of experimental volumetric data (e.g., in .ccp4, .mrc, .map, .rec, .zarr, .ome.tif formats) and advances in segmentation software (e.g., Amira, Segger, IMOD) and formats (e.g., .am, .seg, .mod, etc.) have led to a demand for efficient web-based visualization tools. Despite this, current solutions remain scarce, hindering data interpretation and dissemination. Previously, we introduced Mol* Volumes &amp; Segmentations (Mol* VS), a web application for the visualization of volumetric, segmentation, and annotation data (e.g., semantically relevant information on biological entities corresponding to individual segmentations such as Gene Ontology terms or PDB IDs). However, this lacked important features such as the ability to edit annotations (e.g., assigning user-defined descriptions of a segment) and seamlessly share visualizations. Additionally, setting up Mol* VS required a substantial programming background. This article presents an updated version, Mol* VS 2.0, that addresses these limitations. As part of Mol* VS 2.0, we introduce the Annotation Editor, a user-friendly graphical interface for editing annotations, and the Volumes &amp; Segmentations Toolkit (VSToolkit) for generating shareable files with visualization data. The outlined protocols illustrate the utilization of Mol* VS 2.0 for visualization of volumetric and segmentation data across various scales, showcasing the progress in the field of molecular complex visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: VSToolkit—setting up and visualizing a user-constructed Mol* VS 2.0 database entry</p><p><b>Basic Protocol 2</b>: VSToolkit—visualizing multiple time frames and volume channels</p><p><b>Support Protocol 1</b>: Example: Adding database entry idr-13457537</p><p><b>Alternate Protocol 1</b>: Local-server-and-viewer—visualizing multiple time frames and volume channels</p><p><b>Support Protocol 2</b>: Addition of database entry custom-tubhiswt</p><p><b>Basic Protocol 3</b>: VSToolkit—visualizing a specific channel and time frame</p><p><b>Basic Protocol 4</b>: VSToolkit—visualizing geometric segmentation</p><p><b>Basic Protocol 5</b>: VSToolkit—visualizing lattice segmentations</p><p><b>Alternate Protocol 2</b>: “Local-server-and-viewer”—visualizing lattice segmentations</p><p><b>Basic Protocol 6</b>: “Local-server-and-viewer”—visualizing multiple volume channels</p><p><b>Support Protocol 3</b>: Deploying a server API</p><p><b>Support Protocol 4</b>: Hosting Mol* viewer with VS extension 2.0</p><p><b>Support Protocol 5</b>: Example: Addition of database entry empiar-11756</p><p><b>Support Protocol 6</b>: Example: Addition of database entry emd-1273</p><p><b>Support Protocol 7</b>: Editing annotations</p><p><b>Support Protocol 8</b>: Addition of database entry idr-5025553</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11627126/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142803784","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}

{"title":"Generate and Analyze Three-Dimensional Dendritic Spine Morphology Datasets With SpineTool Software","authors":"Anita Ustinova,&nbsp;Ekaterina Volkova,&nbsp;Anastasiya Rakovskaya,&nbsp;Daria Smirnova,&nbsp;Olesya Korovina,&nbsp;Ekaterina Pchitskaya","doi":"10.1002/cpz1.70061","DOIUrl":"10.1002/cpz1.70061","url":null,"abstract":"<p>Dendritic spine morphology is associated with the current state of the synapse and neuron, and changes during synaptic plasticity in response to stimulus. At the same time, dendritic spine alterations are reported during various neurodegenerative and neurodevelopmental disorders and other brain states. Accurate and informative analysis of spine shape has an urgent need for studying the synaptic processes and molecular pathways in normal and pathological conditions, and for testing synapto-protective strategies during preclinical studies. Primary neuronal cultures enable high quality imaging of dendritic spines and offer a wide spectrum of accessible experimental manipulations. This article outlines the protocol for isolating, culturing, fluorescent labeling, and imaging of mouse primary hippocampal neurons by three-dimensional (3D) confocal microscopy in a normal state and in conditions of low amyloid toxicity—an in vitro model of Alzheimer's disease. An alternate protocol describes the neuronal morphology analysis using the EGFP expressing neurons in line-M transgenic mouse brain slices. Since the dendritic spines are relatively small structures lying close to the confocal microscope resolution limit, their proper segmentation on the images is challenging. This protocol highlights the image-preprocessing steps, including generation of theoretical point spread function and deconvolution, which enhances resolution and removes noise, thereby enhancing the 3D spine reconstruction results. SpineTool, an open source Python–based script, enables 3D segmentation of dendrites and spines and numerical metric calculation, including key measures, such as spine length, volume, and surface area, with a new feature, the chord length distribution histogram, improving clustering results. SpineTool supports both manual and machine learning spine classification (i.e., mushroom, thin, stubby, filopodia) and automated clustering using k-means and DBSCAN methods. This protocol provides detailed instructions for using SpineTool to analyze and classify dendritic spines in control and experimental groups, enhancing our understanding of spine morphology across different experimental conditions. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Obtaining 3D confocal dendritic spine images of hippocampal neuronal culture in normal state and conditions of low amyloid toxicity</p><p><b>Alternate Protocol</b>: Obtaining confocal dendritic spine images of mice hippocampal neurons from fixed brain slices</p><p><b>Support Protocol</b>: Post-processing deconvolution of confocal images</p><p><b>Basic Protocol 2</b>: Segmentation of dendritic spines with SpineTool</p><p><b>Basic Protocol 3</b>: Spine dataset preparation using SpineTool</p><p><b>Basic Protocol 4</b>: Clustering of dendritic spines with SpineTool</p><p><b>Basic Protocol 5</b>: Machine classification of dendritic spines with SpineTool</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142788194","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}

{"title":"Cryopreservation of Human Adult Ventricular Tissue for the Preparation of Viable Myocardial Slices","authors":"Alessandra M. Lodrini,&nbsp;Esmee J. Groeneveld,&nbsp;Meindert Palmen,&nbsp;Jesper Hjortnaes,&nbsp;Anke M. Smits,&nbsp;Marie-José Goumans","doi":"10.1002/cpz1.70068","DOIUrl":"https://doi.org/10.1002/cpz1.70068","url":null,"abstract":"<p>Living myocardial slices (LMS) are ultrathin sections of adult myocardium that can be maintained in culture. These slices provide a unique platform for studying interactions between cardiomyocytes (CMs), other cardiac cell types, and the extracellular matrix while maintaining the cytoarchitecture and electrical phenotype of CMs over extended periods. Despite their advantages over other cardiac models, LMS have limitations, particularly their reliance on slice quality. The primary factor influencing the quality of the slices is the method used to process the cardiac tissue block. Current methods typically require immediate slice preparation following the excision of the tissue block, which restricts the timing of experiments. To address this limitation, we developed a simple procedure for cryopreserving human adult myocardium, allowing the preparation of LMS at a later stage. The protocol provides a list of required equipment and reagents, as well as a detailed description of the methodology for processing the myocardium and slice preparation. We present typical results demonstrating that cryopreserved human cardiac tissue retains biomass and structural integrity comparable to freshly obtained myocardium. Furthermore, we assessed the LMS derived from both fresh and cryopreserved samples. Histological analysis confirmed the preservation of viability, normal cytomorphology, and gap junctions between CMs in all LMS after 24 h and up to 5 days of culture in the absence of electrical stimulation. Cryopreservation extends the interval between tissue collection and LMS preparation, facilitating longer-term and more complex experiments. Further research into the impact of cryopreservation on various cardiac cell types will promote better donor organ management and efficient banking of cardiac samples from a multitude of donors and disease states. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation and preservation of human adult myocardium</p><p><b>Basic Protocol 2</b>: Preparation of adult living myocardial slices from cryopreserved blocks</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70068","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142764068","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}

{"title":"Searching and Using MobiDB Resource 6 to Explore Predictions and Annotations for Intrinsically Disordered Proteins.","authors":"Maria Cristina Aspromonte, Federica Quaglia, Alexander Miguel Monzon, Damiano Clementel, Alessio Del Conte, Damiano Piovesan, Silvio C E Tosatto","doi":"10.1002/cpz1.70077","DOIUrl":"10.1002/cpz1.70077","url":null,"abstract":"<p><p>Intrinsically disordered proteins (IDPs) make up around 30% of eukaryotic proteomes and play a crucial role in cellular processes and in pathological conditions such as neurodegenerative disorders and cancers. However, IDPs exhibit dynamic conformational ensembles and are often involved in the formation of biomolecular condensates. Understanding the function of IDPs is critical to research in many areas of science. MobiDB is a unique resource that serves as a comprehensive knowledgebase of IDPs and intrinsically disordered regions (IDRs), combining disorder annotations from experimental evidence and predictions for a broad range of protein sequences. Over the past decade, MobiDB has evolved with a focus on expanding annotation coverage, standardizing annotation provenance, and enhancing database accessibility. The latest MobiDB, version 6, released in July 2024, includes significant improvements, such as the integration of AlphaFoldDB predictions and a new homology transfer pipeline that has substantially increased the number of entries with high-quality annotations. The user interface has also been updated, highlighting annotation features, clarifying the entry page, and providing an immediate overview of disorder, binding, and disorder functions information in the protein sequence. This protocol guides the user through applications of the MobiDB, including disorder prediction, curated data analysis, and exploration of interaction data. This guide covers how to perform a search in MobiDB annotations using the web interface and the MobiDB REST API for programmatic access. The protocols use a step-by-step walkthrough using the human growth hormone receptor to demonstrate MobiDB's functions for visualization and interpretation of protein disorder data. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Searching MobiDB query formats Basic Protocol 2: Searching MobiDB selected datasets and selected proteomes Basic Protocol 3: Performing a search on the Statistics page in MobiDB Support Protocol: Programmatic access with MobiDB REST API Basic Protocol 4: Visualizing and interpreting a MobiDB Entry: The GHR use case.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":"e70077"},"PeriodicalIF":0.0,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11668231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883994","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}

{"title":"Using Vesicular Stomatitis Virus as a Platform for Directed Protease Evolution.","authors":"Francesco Costacurta, Stefanie Rauch, Dorothee von Laer, Emmanuel Heilmann","doi":"10.1002/cpz1.70074","DOIUrl":"10.1002/cpz1.70074","url":null,"abstract":"<p><p>Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain-of-function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS-CoV-2, MERS-CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using VSV as a platform for directed protease evolution Alternate Protocol: Dose response assay with TCID₅₀ readout Support Protocol 1: A pipeline for high-throughput VSV sequencing Support Protocol 2: Rescue of VSV.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":"e70074"},"PeriodicalIF":0.0,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11664493/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879210","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}

{"title":"Employment of a Newly Defined In Vitro Fertilization Protocol to Determine the Cytoskeletal Machinery, DNA Damage, and Subsequent DNA Repair Resulting from Endocrine Disruption by Hexavalent Chromium in Rat Metaphase II Oocytes.","authors":"Liga Wuri, Paul W Zarutskie, Joe A Arosh, Sakhila K Banu","doi":"10.1002/cpz1.70060","DOIUrl":"https://doi.org/10.1002/cpz1.70060","url":null,"abstract":"<p><p>These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing in vitro fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo EDC treatment of rats and immunostaining of treated oocytes Basic Protocol 2: In vitro fertilization and immunostaining of early-stage embryos.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":"e70060"},"PeriodicalIF":0.0,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879207","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}

{"title":"CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology-Directed Repair Mutagenesis","authors":"Martina Bamundo,&nbsp;Sara Palumbo,&nbsp;Ludovica D'Auria,&nbsp;Caterina Missero,&nbsp;Daniela Di Girolamo","doi":"10.1002/cpz1.70056","DOIUrl":"10.1002/cpz1.70056","url":null,"abstract":"<p>Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by <i>in vitro</i> differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan <i>in vitro</i>, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Knock-out generation by indel mutation in primary human keratinocytes using nucleofection of ribonucleoprotein (RNP) complex</p><p><b>Basic Protocol 2</b>: Deletion of specific genomic region using RNPs via nucleofection</p><p><b>Basic Protocol 3</b>: Use of homology-directed repair system to introduce site-specific mutations</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70056","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142735413","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}

{"title":"Cover Image, Volume 4, Issue 11","authors":"","doi":"10.1002/cpz1.70066","DOIUrl":"https://doi.org/10.1002/cpz1.70066","url":null,"abstract":"<p>The cover image is based on the Article <i>PhyKIT: A Multitool for Phylogenomics</i> by Jacob L. Steenwyk et al., https://doi.org/10.1002/cpz1.70016.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70066","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142737381","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}