Cold Spring Harbor protocols最新文献_第6页

Fluorescent In Situ Hybridization Chain Reaction for RNA in the Drosophila Embryonic and Larval Central Nervous System. 果蝇胚胎和幼虫中枢神经系统 RNA 的荧光原位杂交连锁反应。

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.prot108423

Jake E Henderson, Chris C Wreden, Ellie S Heckscher

{"title":"Fluorescent In Situ Hybridization Chain Reaction for RNA in the Drosophila Embryonic and Larval Central Nervous System.","authors":"Jake E Henderson, Chris C Wreden, Ellie S Heckscher","doi":"10.1101/pdb.prot108423","DOIUrl":"https://doi.org/10.1101/pdb.prot108423","url":null,"abstract":"In the Drosophila nerve cord, much is known about the generation of neurons from neuronal stem cells. Over the lifetime of a neuron, the cumulative expression of genes within that neuron determines its fate. Furthermore, gene expression in mature neurons determines their functional characteristics. It is therefore useful to visualize neural gene expression, which is often done via staining with antibodies to a protein of interest. In cases where there is no antibody to a desired gene product, or when it is useful to detect RNA rather than protein products, fluorescent in situ hybridization chain reaction for RNA (HCR RNA-FISH, or HCR for this protocol) can be used to detect and quantify RNA expression. RNA molecules reside predominantly in the cell soma, so HCR can facilitate determining neuron identity because somata position within the nerve cord is stereotyped across animals. HCR provides high-amplitude, high-fidelity signals. In principle, HCR can be broken down into a detection/hybridization stage and an amplification stage. During detection/hybridization, a probe set hybridizes to multiple sequences within a target gene. In the amplification step, concatemerized fluorescent hairpins bind to the hybridized probes. This two-step process increases the specificity of the fluorescent signal and helps reduce the likelihood of background fluorescence compared to traditional in situ hybridization techniques where the hybridizing probe itself contains the fluorescent signal. Here, we describe a protocol for using HCR to study gene expression in the Drosophila embryonic and larval nerve cord. We also describe how to combine HCR with immunofluorescence staining.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281499","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

Single-Neuron Labeling in Drosophila Using Multicolor FLP-Out. 利用多色 FLP-Out 对果蝇的单神经元进行标记

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.prot108422

Zarion D Marshall, Chris C Wreden, Ellie S Heckscher

{"title":"Single-Neuron Labeling in Drosophila Using Multicolor FLP-Out.","authors":"Zarion D Marshall, Chris C Wreden, Ellie S Heckscher","doi":"10.1101/pdb.prot108422","DOIUrl":"https://doi.org/10.1101/pdb.prot108422","url":null,"abstract":"Neurons exhibit some of the most striking examples of morphological diversity of any cell type. Thus, when studying neurons, the morphology of each neuron must be considered individually. However, neurons densely populate the central nervous system (CNS), making it difficult to ascertain fine morphological features due to a lack of spatial resolution. In Drosophila, this problem can be partially resolved by using driver lines that express the yeast transcription factor GAL4 in subsets of neurons. GAL4 can activate the expression of other introduced genetic elements such as genes for fluorescent proteins or other markers under the control of the GAL4 upstream activation sequences (UAS effectors). However, even highly specific GAL4 lines often label sets of potentially morphologically heterogeneous neurons. Here, we describe a protocol for using the multicolor flip-out (MCFO) technique in Drosophila melanogaster to stochastically label individual neurons within a GAL4 expression pattern. MCFO relies on the binary GAL4/UAS expression system in Drosophila but adds additional control for how densely the neurons within a GAL4 expression pattern are labeled via user-controlled heat shock. Specifically, three discrete UAS effector elements containing the sequences for unique epitope tags (FLAG, HA, and V5) linked to a gene for nonfluorescent GFP can be independently expressed under the control of GAL4 only when a transcriptional stop sequence in the UAS promoter sequence has been removed by heat shock-induced recombination. This effectively labels multiple individual neurons with either one or a combination of epitope tags that can be spectrally resolved with immunofluorescence. The MCFO technique is ideal for researchers who want to determine morphological features of CNS neurons in wild-type or mutant backgrounds.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281503","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

Imaging Neural Activity in Intact, Semirestrained Drosophila Larvae. 完整、半受约束果蝇幼体的神经活动成像

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.prot108421

Deeptha Vasudevan, Chris C Wreden, Ellie S Heckscher

{"title":"Imaging Neural Activity in Intact, Semirestrained Drosophila Larvae.","authors":"Deeptha Vasudevan, Chris C Wreden, Ellie S Heckscher","doi":"10.1101/pdb.prot108421","DOIUrl":"https://doi.org/10.1101/pdb.prot108421","url":null,"abstract":"The Drosophila larval nerve cord, which is the equivalent of the vertebrate spinal cord, houses the circuits required to process somatosensory stimuli (e.g., tactile, temperature, vibration, and self-movement) and generate the patterned muscle contractions underlying movement and behavior. Within this complex structure reside many cell types and cellular processes, making it difficult to experimentally access, when compared to peripheral parts of the nervous system (i.e., primary sensory neuron dendrites, motor neuron axons and synapses, and muscles). Additionally, the neurons in the larval nerve cord have small cell bodies, precluding traditional electrophysiological approaches. As such, the function of neurons in the nerve cord is less well studied than other parts of the nervous system, severely limiting our understanding of how larvae process sensory information and generate movement. Ca2+-sensitive fluorescent proteins enable the study of neuronal activity in live, genetically tractable animals, even those with small neuronal cell bodies. In addition, live imaging of neurons within the nerve cord in whole, intact animals is possible because larvae are translucent, and the use of intact animals allows for the peripheral sensory neuron circuits to remain intact. Ca2+-sensitive fluorescent proteins increase their fluorescence when voltage-gated Ca2+ channels are opened in depolarized neurons. Here, we describe an assay where a Ca2+-sensitive fluorescent protein (GCaMP6m) is expressed under the control of a GAL4 driver in a subset of neurons that reside in a circuit for vibration sensation. External vibration (sound) stimulates sensory neurons that activate the cells expressing the Ca2+-sensitive fluorescent protein. Visualization of the calcium-induced fluorescent signal with microscopy allows for quantification of neuronal activity.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281501","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

Use of Maize (Zea mays L.) Mutator Transposon-Induced Mutants of the BonnMu Resource for Forward and Reverse Genetics Studies. 利用波恩姆资源的玉米（Zea mays L.）突变体转座子诱导突变体进行正向和反向遗传学研究。

Cold Spring Harbor protocols Pub Date : 2024-09-09 DOI: 10.1101/pdb.prot108587

Yan Naing Win, Martin Pöschel, Tyll Stöcker, Xuelian Du, Alina Klaus, Ben Wilhelm Braun, Linnéa Lukas, Alexa Brox, Heiko Schoof, Frank Hochholdinger, Caroline Marcon

{"title":"Use of Maize (Zea mays L.) Mutator Transposon-Induced Mutants of the BonnMu Resource for Forward and Reverse Genetics Studies.","authors":"Yan Naing Win, Martin Pöschel, Tyll Stöcker, Xuelian Du, Alina Klaus, Ben Wilhelm Braun, Linnéa Lukas, Alexa Brox, Heiko Schoof, Frank Hochholdinger, Caroline Marcon","doi":"10.1101/pdb.prot108587","DOIUrl":"https://doi.org/10.1101/pdb.prot108587","url":null,"abstract":"The BonnMu resource represents a tagged collection of maize (Zea mays L.) Mutator (Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of the BonnMu collection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire a BonnMu F2 stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation of Mu insertions by genotyping via PCR, with the ultimate goal of establishing genotype-phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281506","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

BonnMu: A Resource for Functional Genomics in Maize (Zea mays L.). BonnMu：玉米功能基因组学资源。

Cold Spring Harbor protocols Pub Date : 2024-09-09 DOI: 10.1101/pdb.top108465

Caroline Marcon, Yan Naing Win, Xuelian Du, Frank Hochholdinger

引用次数: 0

Identification of Transposon Insertion Sites in Maize Mu-Tagged Mutants Using Mu-Seq. 利用 Mu-Seq 鉴定玉米 Mu 标记突变体中的转座子插入位点

Cold Spring Harbor protocols Pub Date : 2024-09-09 DOI: 10.1101/pdb.prot108586

Caroline Marcon, Alexa Brox, Yan Naing Win, Tyll Stöcker, Xuelian Du, Heiko Schoof, Frank Hochholdinger

{"title":"Identification of Transposon Insertion Sites in Maize Mu-Tagged Mutants Using Mu-Seq.","authors":"Caroline Marcon, Alexa Brox, Yan Naing Win, Tyll Stöcker, Xuelian Du, Heiko Schoof, Frank Hochholdinger","doi":"10.1101/pdb.prot108586","DOIUrl":"https://doi.org/10.1101/pdb.prot108586","url":null,"abstract":"Mutator (Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as the BonnMu resource, a Mu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping of Mu insertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) from Mu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location of Mu insertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maize Mu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281500","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

Sampling and Analysis of the Maize Microbiome. 玉米微生物组的取样和分析。

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.top108463

Jason G Wallace, Alonso Favela, Sierra Raglin

引用次数: 0

Navigating the Maze of Maize Genomics: the Impact of Transposable Elements and Tandem Repeats. 玉米基因组学的迷宫导航：可转座元件和串联重复的影响。

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.top108441

Pedro Heringer, Christopher W Benson, Shujun Ou

{"title":"Navigating the Maze of Maize Genomics: the Impact of Transposable Elements and Tandem Repeats.","authors":"Pedro Heringer, Christopher W Benson, Shujun Ou","doi":"10.1101/pdb.top108441","DOIUrl":"https://doi.org/10.1101/pdb.top108441","url":null,"abstract":"Transposable elements (TEs) are abundant and ubiquitous components of eukaryotic genomes. Since TEs were first discovered in maize (Zea mays) by Barbara McClintock in the late 1940s, these elements have been shown to be important agents in shaping genome structure and evolution. Today, maize continues to be an important model organism for molecular and quantitative genetics, and represents a particularly useful system for the study of the interplay between TEs and host genomes. While TEs constitute a significant part of the maize genome and are important drivers of genome evolution, their annotation remains a complex and challenging task. Here, we discuss genome annotation of TEs and other repetitive sequences in maize genomes. We briefly review current knowledge on the overall landscape of TE and non-TE repeats in maize, and discuss how these sequences may impact genome structure, and the genotype and phenotype within species. We also provide a summary of the main tools used to find TE polymorphisms, and briefly introduce four different bioinformatic approaches for TE and tandem repeat annotation, explaining how they can be best used by maize researchers.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142139528","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

Preparation of Illumina 16s Amplicon Sequencing Libraries with Peptide Nucleic Acids (PNAs) for the Analysis of Maize-Associated Microbiomes. 用多肽核酸 (PNA) 制备用于分析玉米相关微生物组的 Illumina 16s 扩增片段测序文库。

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.prot108583

Jason G Wallace, Holly Griffis

引用次数: 0

Sampling Maize (Zea mays) Seed Endophytes. 玉米（Zea mays）种子内生菌采样。

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.prot108582

Jason G Wallace, Daniel Laspisa

引用次数: 0