Bio-protocol最新文献

Live Cell Imaging to Monitor Axonal Pruning in Drosophila Motor Neurons. 活细胞成像监测果蝇运动神经元轴突修剪。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5367

Keyao Long, Wanyue Xu, Xun Miao, Su Wang, Menglong Rui

{"title":"Live Cell Imaging to Monitor Axonal Pruning in Drosophila Motor Neurons.","authors":"Keyao Long, Wanyue Xu, Xun Miao, Su Wang, Menglong Rui","doi":"10.21769/BioProtoc.5367","DOIUrl":"10.21769/BioProtoc.5367","url":null,"abstract":"Over the lifespan of an individual, brain function requires adjustments in response to environmental changes and learning experiences. During early development, neurons overproduce neurite branches, and neuronal pruning removes the unnecessary neurite branches to make a more accurate neural circuit. Drosophila motoneurons prune their intermediate axon bundles rather than the terminal neuromuscular junction (NMJ) by degeneration, which provides a unique advantage for studying axon pruning. The pruning process of motor axon bundles can be directly analyzed by real-time imaging, and this protocol provides a straightforward method for monitoring the developmental process of Drosophila motor neurons using live cell imaging. Key features • Long-range projecting axon bundles of Drosophila motor neurons extending from soma on the ventral nerve cord (VNC) undergo degeneration rather than retraction during metamorphosis. • The pruning process of motor axon bundles can be directly observed by real-time live-cell imaging. • The complete clearance of axon bundles occurs approximately 22 h after pupal formation (22 h APF). • Mushroom body (MB) γ neuron axon pruning regulatory genes are conserved for motor neurons.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5367"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245631/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628091","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

Untargeted Metabolomics of Epimastigote Forms of Trypanosoma cruzi. 克氏锥虫表皮马鞭毛体的非靶向代谢组学研究。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5368

Michel Augusto Silva, Mario Izidoro, Bruno Souza Bonifácio, Sergio Schenkman

{"title":"Untargeted Metabolomics of Epimastigote Forms of Trypanosoma cruzi.","authors":"Michel Augusto Silva, Mario Izidoro, Bruno Souza Bonifácio, Sergio Schenkman","doi":"10.21769/BioProtoc.5368","DOIUrl":"10.21769/BioProtoc.5368","url":null,"abstract":"Trypanosoma cruzi, the causative agent of Chagas disease, faces significant metabolic challenges due to fluctuating nutrient availability and oxidative stress within its insect vector. Metabolomic techniques, such as gas chromatography-mass spectrometry (GC-MS), have been widely used to study the adaptive mechanisms of the parasite. This article describes a standardized method for the untargeted metabolomics analysis of T. cruzi epimastigote, covering parasite cultivation, sample deproteinization with methanol, metabolite extraction, derivatization with BSTFA, and GC-MS analysis. To ensure robustness and reproducibility, statistical analysis uses univariate tests, as well as multivariate approaches such as principal component analysis (PCA) and partial least squares (PLS) regression. The protocol offers a reliable and sensitive method to study metabolic responses in T. cruzi under environmental stress, with low biological variability and high reproducibility. Key features • GC-MS was used to conduct a standardized metabolomics investigation of Trypanosoma cruzi epimastigote, assuring reproducibility and minimum biological variability. • Includes sample deproteinization, metabolite extraction, and derivatization with BSTFA for accurate metabolite profiling under different biological conditions. • Employs robust statistical approaches (PCA, PLS) to investigate differences among experimental groups and detect significant alterations in metabolism. • Internal standards and multiple replicates ensure high sensitivity and repeatability, which is excellent for investigating metabolic processes in protozoan parasites.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5368"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245627/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628097","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

An Alternative Gene Editing Strategy Using a Single AAV Vector. 使用单个AAV载体的替代基因编辑策略。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5362

Jamie C Moffa, Vani Kalyanaraman, Bryan A Copits

{"title":"An Alternative Gene Editing Strategy Using a Single AAV Vector.","authors":"Jamie C Moffa, Vani Kalyanaraman, Bryan A Copits","doi":"10.21769/BioProtoc.5362","DOIUrl":"10.21769/BioProtoc.5362","url":null,"abstract":"We recently developed an approach for cell type-specific CRISPR/Cas9 editing and transgene expression using a single viral vector. Here, we present a protocol describing how to design and generate plasmids and adeno-associated viruses (AAVs) compatible with this single-vector gene editing approach. This protocol has four components: (1) guide RNA (gRNA) design to target specific genes of interest, (2) ligation and cloning of CRISPR-competent AAV vectors, (3) production of vector-containing AAVs, and (4) viral titer quantification. The resultant vectors are compatible for use with mouse lines expressing the Cas9 protein from Streptococcus pyogenes (SpCas9) and Cre recombinase to enable selective co-expression of standard neuroscience tools in edited cells. This protocol can produce AAVs of any serotype, and the resulting AAVs can be used in the central and peripheral nervous systems. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection. Key features • Single-vector CRISPR/Cas9 gene editing and genetically encoded tool delivery for use in mouse central and peripheral nervous systems. • Can be combined with many genetically encoded tools, including fluorescent proteins, optogenetic and chemogenetic tools, and calcium imaging. • Requires first-generation cross between a Cre driver mouse line and Cre-dependent SpCas9 mouse line. • Optimized for use with SpCas9.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5362"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12242552/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628082","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

Isolation of Mitochondria From Yeast to Estimate Mitochondrial Pools of Inorganic Phosphate. 酵母线粒体的分离及其对无机磷酸盐池的估计。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5370

Swagata Adhikary, Vineeth Vengayil, Sunil Laxman

{"title":"Isolation of Mitochondria From Yeast to Estimate Mitochondrial Pools of Inorganic Phosphate.","authors":"Swagata Adhikary, Vineeth Vengayil, Sunil Laxman","doi":"10.21769/BioProtoc.5370","DOIUrl":"10.21769/BioProtoc.5370","url":null,"abstract":"Mitochondria are dynamic organelles with essential roles in energetics and metabolism. Several metabolites are common to both the cytosolic and mitochondrial fractions of the cell. The compartmentalization of metabolites within the mitochondria allows specialized uses for mitochondrial metabolism. Inorganic phosphate (Pi) is one such critical metabolite required for ATP synthesis, via glycolysis and mitochondrial oxidative phosphorylation. Estimating total cellular Pi levels cannot distinguish the distribution of Pi pools across different cellular compartments, such as the cytosol and mitochondria, and therefore separate the contributions made toward glycolysis or other cytosolic metabolic processes vs. mitochondrial outputs. Quantifying Pi pools in mitochondria can therefore be very useful toward understanding mitochondrial metabolism and phosphate homeostasis. Here, we describe a protocol for the fairly rapid, efficient isolation of mitochondria from Saccharomyces cerevisiae by immunoprecipitation for quantitative estimation of mitochondrial and cytosolic Pi pools. This method utilizes magnetic beads to capture FLAG-tagged mitochondria (Tom20-FLAG) from homogenized cell lysates. This method provides a valuable tool to investigate changes in mitochondrial phosphate dynamics. Additionally, this protocol can be coupled with LC-MS approaches to quantitatively estimate mitochondrial metabolites and proteins and can be similarly used to assess other metabolite pools that are partitioned between the cytosol and mitochondria. Key features • This protocol describes how to isolate mitochondria from Saccharomyces cerevisiae for quantitative estimation of inorganic phosphate or other metabolites. • Mitochondria are efficiently isolated by immunoprecipitation using magnetic beads, bypassing the need for time-consuming density-based centrifugation. • This method can be integrated into LC-MS-based workflows to quantify mitochondrial metabolites and proteins.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5370"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245623/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628088","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

Protocol for Generation of Single-Gene Knockout in Hard-to-Transfect THP1 Cell Lines Using CRISPR/Cas9. 使用CRISPR/Cas9在难以转染的THP1细胞系中产生单基因敲除的方案。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5365

Kaveri Srivastava, Bhaswati Pandit

{"title":"Protocol for Generation of Single-Gene Knockout in Hard-to-Transfect THP1 Cell Lines Using CRISPR/Cas9.","authors":"Kaveri Srivastava, Bhaswati Pandit","doi":"10.21769/BioProtoc.5365","DOIUrl":"10.21769/BioProtoc.5365","url":null,"abstract":"This protocol provides a step-by-step approach for generating single-gene knockout in hard-to-transfect suspension immune cell lines like THP1, specifically demonstrated by knocking out the GSDMD gene. By employing CRISPR-Cas9 system delivered via lentivirus, this protocol enables precise gene disruption through targeted single-guide RNAs (sgRNAs). Key steps include designing specific sgRNAs, cloning them into a CRISPR vector, viral packaging, and transducing the target cells, followed by selection and validation. This optimized protocol is particularly useful for functional studies in immune cells, allowing researchers to reliably explore gene function in complex cellular pathways. Key features • CRISPR-Cas9-based knockout strategy tailored for hard-to-transfect THP1 cells using lentiviral delivery. • Lentiviral transduction ensures stable gene delivery with high efficiency compared to other traditional methods like transfection and electroporation. • Stepwise validation through colony PCR, sequencing, and western blotting to confirm successful knockout. • Scalable approach, applicable to various cell models for functional genomic studies. Graphical overview.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5365"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245624/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628092","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

RACE-Nano-Seq: Profiling Transcriptome Diversity of a Genomic Locus. RACE-Nano-Seq：基因组位点转录组多样性分析。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5374

Lu Tang, Dongyang Xu, Philipp Kapranov

{"title":"RACE-Nano-Seq: Profiling Transcriptome Diversity of a Genomic Locus.","authors":"Lu Tang, Dongyang Xu, Philipp Kapranov","doi":"10.21769/BioProtoc.5374","DOIUrl":"10.21769/BioProtoc.5374","url":null,"abstract":"The complexity of the human transcriptome poses significant challenges for complete annotation. Traditional RNA-seq, often limited by sensitivity and short read lengths, is frequently inadequate for identifying low-abundant transcripts and resolving complex populations of transcript isoforms. Direct long-read sequencing, while offering full-length information, suffers from throughput limitations, hindering the capture of low-abundance transcripts. To address these challenges, we introduce a targeted RNA enrichment strategy, rapid amplification of cDNA ends coupled with Nanopore sequencing (RACE-Nano-Seq). This method unravels the deep complexity of transcripts containing anchor sequences-specific regions of interest that might be exons of annotated genes, in silico predicted exons, or other sequences. RACE-Nano-Seq is based on inverse PCR with primers targeting these anchor regions to enrich the corresponding transcripts in both 5' and 3' directions. This method can be scaled for high-throughput transcriptome profiling by using multiplexing strategies. Through targeted RNA enrichment and full-length sequencing, RACE-Nano-Seq enables accurate and comprehensive profiling of low-abundance transcripts, often revealing complex transcript profiles at the targeted loci, both annotated and unannotated. Key features • This protocol is highly sensitive and can detect low-abundance transcripts. • This protocol can be performed in a typical molecular biology laboratory. • This protocol allows RACE reactions with single or multiple primers, supporting various research scales. • This protocol enables characterization of complex genomic loci and discovery of novel transcripts, exons, and alternative splicing events.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5374"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245629/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628094","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

Derivation and Culture of Enriched Phrenic-Like Motor Neurons From Human iPSCs. 从人多能干细胞中衍生和培养富集膈样运动神经元。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5363

Louise Thiry, Julien Sirois, Thomas M Durcan, Stefano Stifani

{"title":"Derivation and Culture of Enriched Phrenic-Like Motor Neurons From Human iPSCs.","authors":"Louise Thiry, Julien Sirois, Thomas M Durcan, Stefano Stifani","doi":"10.21769/BioProtoc.5363","DOIUrl":"10.21769/BioProtoc.5363","url":null,"abstract":"The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm, leading to death by respiratory failure. Human experimental models to study phMNs are lacking, hindering the understanding of the mechanisms of phMN degeneration in ALS. Here, we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development, phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels, under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine, followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research, it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest. Key features • This protocol generates enriched hiPSC-derived phrenic motor neuron cultures. • The protocol can be used to develop models to study human respiratory motor neuron disease. • The protocol allows the generation of phrenic motor neuron preparations with potential for motor neuron replacement strategies. • The protocol requires experience in hiPSC culturing and FACS-based cell sorting for a successful outcome.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5363"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12242553/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628086","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

Isolation of Podocyte Cell Fractions From Mouse Kidney Using Magnetic Activated Cell Sorting (MACS). 磁活化细胞分选（MACS）分离小鼠肾足细胞组分。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5364

Jeffrey W Pippin, Carol J Loretz, Diana G Eng, Oliver Wessely, Stuart J Shankland

{"title":"Isolation of Podocyte Cell Fractions From Mouse Kidney Using Magnetic Activated Cell Sorting (MACS).","authors":"Jeffrey W Pippin, Carol J Loretz, Diana G Eng, Oliver Wessely, Stuart J Shankland","doi":"10.21769/BioProtoc.5364","DOIUrl":"10.21769/BioProtoc.5364","url":null,"abstract":"Glomerular diseases characterized by injury to post-mitotic epithelial cells called podocytes are a leading cause of chronic kidney disease. Yet, isolating podocytes from the kidney for transcriptomic, proteomic, and metabolomic studies has been a major technical challenge. Protocols utilizing glomerular sieving and laser capture methods are of limited use because they are not podocyte-specific but instead capture all four glomerular cell types. Here, we present a magnetic-activated cell sorting (MACS) method where podocytes are isolated from digested whole kidneys using antibodies specific to extracellular antigens on podocytes. Using microbeaded secondary antibodies binding to the podocyte-specific primary antibodies allows sorting of the podocytes using a magnet. This podocyte-only cell fraction is a unique source of in vivo-derived cells for molecular and cellular experiments. Key features • The protocol isolates a podocyte-only cell population from kidneys that is readily available for molecular and cellular studies. • The non-podocyte fraction serves as a matching negative control. • High cell yields are obtained. • The method can be applied to separately isolate podocytes from the outer cortex and juxtamedullary regions of the kidney.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5364"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12242555/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628089","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

Assessing the Efficiency of Double-Strand Break Repair Mediated by Homologous Recombination and Non-homologous End-Joining Pathways in Saccharomyces cerevisiae. 酿酒酵母同源重组和非同源末端连接途径介导的双链断裂修复效率评估。

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5378

Sugith Badugu, Kshitiza Mohan Dhyani, Kalappa Muniyappa

{"title":"Assessing the Efficiency of Double-Strand Break Repair Mediated by Homologous Recombination and Non-homologous End-Joining Pathways in Saccharomyces cerevisiae.","authors":"Sugith Badugu, Kshitiza Mohan Dhyani, Kalappa Muniyappa","doi":"10.21769/BioProtoc.5378","DOIUrl":"10.21769/BioProtoc.5378","url":null,"abstract":"The DNA double-strand breaks (DSBs) generated by exogenous and endogenous factors are repaired by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). These two pathways compete for DSB repair, and the choice of pathway depends on the context of the DNA lesion, the stage of the cell cycle, and the ploidy in the yeast Saccharomyces cerevisiae. However, the mechanistic details of the DSB repair pathway choice and its consequences for S. cerevisiae genome stability remain unclear. Here, we present PCR-based and cell-based assays as well as data analysis methods to quantitatively measure the efficiency of HR and NHEJ at DSBs in S. cerevisiae. An intermolecular recombination assay between plasmid and chromosomal DNA involving G-quadruplex DNA and a \"suicide-deletion\" assay have been utilized to evaluate the efficiency of HR and NHEJ, respectively. These streamlined protocols and optimized growth conditions can be used to identify the NHEJ- and HR-deficient S. cerevisiae mutant strains. Key features • Optimized protocol for intermolecular recombination involving G-quadruplex-forming DNA sequences derived from recombination hotspots in S. cerevisiae. • Optimized protocol to quantify the efficiency of NHEJ in S. cerevisiae. • Quantitative assessment of HR and NHEJ efficiency and data validation. This protocol is used in: eLife (2024), DOI: 10.7554/eLife.96933.3.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5378"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245625/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628084","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

APEX2 RNA Proximity Labeling in Mammalian Cell Lines With Low Biotin Permeability. 低生物素渗透性哺乳动物细胞系的APEX2 RNA邻近标记

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Bio-protocol Pub Date : 2025-07-05 DOI: 10.21769/BioProtoc.5372

Adrian Beat Tschan, Arpan Kumar Rai, Lucas Pelkmans, Alexa B R McIntyre

{"title":"APEX2 RNA Proximity Labeling in Mammalian Cell Lines With Low Biotin Permeability.","authors":"Adrian Beat Tschan, Arpan Kumar Rai, Lucas Pelkmans, Alexa B R McIntyre","doi":"10.21769/BioProtoc.5372","DOIUrl":"10.21769/BioProtoc.5372","url":null,"abstract":"The subcellular localization of RNA plays a critical role in various biological processes, including development and stress response. Proximity labeling eases the detection of localized transcripts and protein enrichment compared to previous techniques that rely on biochemical isolation of subcellular structures. The rapid reaction and small labeling radius of APEX2 make it an attractive alternative to other proximity labeling approaches, such as BioID. However, we found that standard protocols for APEX proximity labeling fail in human induced pluripotent stem cells. Moreover, standard protocols yield heterogeneous labeling of biomolecules across single cells in MCF10A breast epithelial cells. Our results indicate that low biotin permeability in these cell lines is the main cause for failed or inefficient labeling. This protocol outlines improved labeling by combining the rapid hydrogen peroxide-driven APEX2 reaction with the addition of a mild detergent during biotin incubation. This adaptation leads to efficient proximity labeling in hiPSCs and more homogeneous biotinylation across single cells in MCF10As. The adapted protocol extends the use of APEX2 proximity labeling to cell lines with poor biotin permeability. Key features • Builds on methods developed by the Ting Lab [1] and the Ingolia Lab [2] for proximity labeling of transcripts using the enzyme APEX2. • Focuses on cell lines with low biotin permeability, like human induced pluripotent stem cells, by including a mild detergent to increase biotin uptake. • Includes controls for nonspecific molecular localization and statistical methods for processing of resulting sequencing data.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 13","pages":"e5372"},"PeriodicalIF":1.0,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12245634/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628083","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