Methods in cell biology最新文献_第7页

A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine. 用于监测低甲基化胞嘧啶类似物地西他滨和阿扎胞苷治疗的药效学试验。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-21 DOI: 10.1016/bs.mcb.2024.02.016

James W Jacobberger, Philip G Woost

引用次数: 0

Spectral analysis and sorting of microbial organisms using a spectral sorter. 使用光谱分拣机对微生物进行光谱分析和分拣。

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Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-12 DOI: 10.1016/bs.mcb.2024.02.017

Sharath Narayana Iyengar, J Paul Robinson

引用次数: 0

Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation. 通过中心粒失活诱导染色体特异性微核和染色体三分裂。

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Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-11-28 DOI: 10.1016/bs.mcb.2022.10.009

Yu-Fen Lin, Qing Hu, Alison Guyer, Daniele Fachinetti, Peter Ly

{"title":"Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation.","authors":"Yu-Fen Lin, Qing Hu, Alison Guyer, Daniele Fachinetti, Peter Ly","doi":"10.1016/bs.mcb.2022.10.009","DOIUrl":"10.1016/bs.mcb.2022.10.009","url":null,"abstract":"Chromothripsis describes the catastrophic fragmentation of individual chromosomes followed by its haphazard reassembly into a derivative chromosome harboring complex rearrangements. This process can be initiated by mitotic cell division errors when one or more chromosomes aberrantly mis-segregate into micronuclei and acquire extensive DNA damage. Approaches to induce the formation of micronuclei encapsulating random chromosomes have been used; however, the eventual reincorporation of the micronucleated chromosome into daughter cell nuclei poses a challenge in tracking the chromosome for multiple cell cycles. Here we outline an approach to genetically engineer stable human cell lines capable of efficient chromosome-specific micronuclei induction. This strategy, which targets the CENP-B-deficient Y chromosome centromere for inactivation, allows the stepwise process of chromothripsis to be experimentally recapitulated, including the mechanisms and timing of chromosome fragmentation. Lastly, we describe the integration of a selection marker onto the micronucleated Y chromosome that enables the diverse genomic rearrangement landscape arising from micronuclei formation to be interrogated.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"182 ","pages":"1-20"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11008423/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino. 通过脑室显微注射 vivo morpholino 在斑马鱼幼体中生成胶质细胞源性神经营养因子 (gdnf) 形态体。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-10-31 DOI: 10.1016/bs.mcb.2022.09.004

Suzita Mohd Noor, Chee Ern David Wong, Pooi-Fong Wong, Anwar Norazit

{"title":"Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.","authors":"Suzita Mohd Noor, Chee Ern David Wong, Pooi-Fong Wong, Anwar Norazit","doi":"10.1016/bs.mcb.2022.09.004","DOIUrl":"10.1016/bs.mcb.2022.09.004","url":null,"abstract":"Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"181 ","pages":"17-32"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Methods for detection of mitochondrial reactive oxygen species in senescent cells. 检测衰老细胞线粒体活性氧的方法。

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Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-10-14 DOI: 10.1016/bs.mcb.2022.09.011

Fenniche Salma, Oubaddou Yassire, Bakri Youssef, Dupuy Corinne, Rabii Ameziane El Hassani

{"title":"Methods for detection of mitochondrial reactive oxygen species in senescent cells.","authors":"Fenniche Salma, Oubaddou Yassire, Bakri Youssef, Dupuy Corinne, Rabii Ameziane El Hassani","doi":"10.1016/bs.mcb.2022.09.011","DOIUrl":"10.1016/bs.mcb.2022.09.011","url":null,"abstract":"Cellular senescence is a pathophysiological process with multifaceted effects. It is involved in wound healing, aging and age-related diseases as well as cancer. On the one hand, senescence is considered as barrier against tumorigenesis by inducing an irreversible/prolonged cell cycle arrest. On the other hand, it may promote tumorigenesis when senescent cells accumulate genomic instability and bypass this cell cycle arrest. Interestingly, the bystander effects mediate the propagation of the genetic instability from senescent cells to their environment through the SASP (Senescence Associated Secretory Phenotype) including proinflammatory cytokines, proteases, growth factors and Reactive Oxygen Species 'ROS.' From several markers explored to detect senescent cells (β-galactosidase, p16, p21, p53, heterochromatin foci, DNA damage,…), ROS arouse particular interest because of their involvement at the chronic supraphysiological level, in the induction and maintain of DNA damage, inflammation, cell cycle disruption and epigenetic instability. In this context, the choice of methods to detect ROS in senescent cells is of particular interest and must take into account relevant parameters as well as the specificity for each species of ROS and the subcellular localization of ROS production. In this chapter, we introduce senescence and ROS, we briefly discuss the advantages and the shortcomings of methods routinely used to detect ROS. In addition, we describe the protocol to detect ROS at mitochondrial level (using the MitoSOX staining) in the BCPAP cell line (from human papillary thyroid carcinomas) expressing BRAFV600E oncogene known to trigger senescence.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"181 ","pages":"33-41"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions. 基于高浓度流式细胞仪和 CRISPR-Cas9 双平台，量化基因相互作用。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-03-25 DOI: 10.1016/bs.mcb.2023.02.005

Natasha Ramakrishnan, Taylor Malachowski, Priyanka Verma

{"title":"A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions.","authors":"Natasha Ramakrishnan, Taylor Malachowski, Priyanka Verma","doi":"10.1016/bs.mcb.2023.02.005","DOIUrl":"10.1016/bs.mcb.2023.02.005","url":null,"abstract":"Probing epistasis between two genes can be a critical first step in identifying the molecular players in a cellular pathway. The advent of CRISPR-Cas mediated genetic screen has enabled studying of these genetic interactions at a genomic scale. However, when combining depletion of two genes using CRISPR Cas9, reduced targeting efficiencies due to competition for Cas loading and recombination in the cloning step have emerged as key challenges. Moreover, given conventional CRISPR screens typically involve comparison between the initial and final time point, it is difficult to parse the time kinetics with which a perturbed genetic interaction impacts viability, and it also becomes challenging to assess epistasis with essential genes. Here, we discuss a high-throughput flow-based approach to study genetic interactions. By utilizing two different Cas9 orthologs and monitoring viability at multiple time points, this approach helps to effectively mitigate the limitations of Cas9 competition and enables assessment of genetic interactions with both essential and non-essential genes at a high temporal resolution.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"182 ","pages":"299-312"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An automated image analysis pipeline to quantify the coordination and overlap of transcription and replication activity in mammalian genomes. 用于量化哺乳动物基因组中转录和复制活动的协调与重叠的自动图像分析管道。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-07-03 DOI: 10.1016/bs.mcb.2023.05.012

Maxime Lalonde, Henning Ummethum, Manuel Trauner, Andreas Ettinger, Stephan Hamperl

{"title":"An automated image analysis pipeline to quantify the coordination and overlap of transcription and replication activity in mammalian genomes.","authors":"Maxime Lalonde, Henning Ummethum, Manuel Trauner, Andreas Ettinger, Stephan Hamperl","doi":"10.1016/bs.mcb.2023.05.012","DOIUrl":"10.1016/bs.mcb.2023.05.012","url":null,"abstract":"Transcription-replication conflicts (TRCs) represent a potent endogenous source of replication stress. Besides the spatial and temporal coordination of replication and transcription programs, cells employ many additional mechanisms to resolve TRCs in a timely manner, thereby avoiding replication fork stalling and genomic instability. Proximity ligation assays (PLA) using antibodies against actively elongating RNA Polymerase II (RNAPIIpS2) and PCNA to detect proximity (<40nm) between transcribing RNA polymerases and replication forks can be used to assess and quantify TRC levels in cells. A complementary fluorescence microscopy approach to assess the spatial coordination of transcription and replication activities in the nucleus is to quantify the colocalization (200-400nm) between active transcription and ongoing replication using immunofluorescence staining with an antibody against elongating RNA Polymerase II (RNAPIIpS2) and EdU-Click-it pulse-labelling, respectively. Despite significant efforts to automate image analysis, the need for manual verification, correction, and complementation of automated processes creates a bottleneck for efficient, high-throughput and large-scale imaging. Here, we describe an automated Fiji image analysis macro that allows the user to automate the measurement of RNAPIIpS2 and EdU levels and extract the key parameters such as transcription-replication (TR) colocalization and TRC-PLA foci count from single cells in a high throughput manner. While we showcase the usability of this analysis pipeline for quantifying TR colocalization and TRC-PLA in mouse embryonic stem cells (mESCs), the analysis pipeline is designed as a generally applicable tool allowing the quantification of nuclear signals, colocalization and foci count in various model systems and cell types.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"182 ","pages":"199-219"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enrichment of DNA replication intermediates by EdU pull down. 通过 EdU 牵引富集 DNA 复制中间体。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-12-13 DOI: 10.1016/bs.mcb.2022.11.001

Fabio Pessina, Alessia Romussi, Daniele Piccini, Giulia Mazzucco, Mario Varasi, Ylli Doksani

引用次数: 0

Mapping histone variant genomic distribution: Exploiting SNAP-tag labeling to follow the dynamics of incorporation of H3 variants. 组蛋白变体基因组分布图：利用 SNAP 标记跟踪 H3 变体的整合动态。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-12-05 DOI: 10.1016/bs.mcb.2022.10.007

Audrey Forest, Jean-Pierre Quivy, Geneviève Almouzni

{"title":"Mapping histone variant genomic distribution: Exploiting SNAP-tag labeling to follow the dynamics of incorporation of H3 variants.","authors":"Audrey Forest, Jean-Pierre Quivy, Geneviève Almouzni","doi":"10.1016/bs.mcb.2022.10.007","DOIUrl":"10.1016/bs.mcb.2022.10.007","url":null,"abstract":"In the eukaryotic cell nucleus, in addition to the genomic information, chromatin organization provides an additional set of information which is more versatile and associates with distinct cell identities. In particular, the marking of the nucleosomes by a choice of specific histone variants can potentially confer distinct functional properties critical for genome function and stability. To understand how this unique marking operates we need to access to the genomic distribution of each variant. A general approach based on ChIP-Seq, relies on the specific isolation of DNA bound to the variant of interest, usually using cross-linked material and specific antibodies. The availability of reliable specific antibodies recognizing with high affinity crosslinked antigen represents a limitation. Here, we describe an experimental approach exploiting a tag fused to the protein of interest. The chose protein is a histone variant and we use native conditions for the selective capture of the histone variant in a nucleosome. Most importantly, we describe how to use a particular labeling system, with a SNAP tag enabling to specifically capture nucleosomes comprising newly synthesized histones. This method allows to follow the newly deposited histone variant at various times thereby offering a unique opportunity to evaluate the dynamics of histone deposition genome wide. We describe the method here for H3 variant, but it can be adapted to any histone variant with the appropriate fused tag to address genome wide a turn-over associated to the biological context of interest.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"182 ","pages":"49-65"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Spectral flow cytometry: Fundamentals and future impact. 光谱流式细胞仪：基本原理和未来影响。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-04-04 DOI: 10.1016/bs.mcb.2024.02.022

J Paul Robinson, Bartek Rajwa

引用次数: 0