Cold Spring Harbor protocols最新文献_第2页

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 : 2025-09-02 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":"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":"pdb.prot108586"},"PeriodicalIF":0.0,"publicationDate":"2025-09-02","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

Four Strategies for Whole-Genome Annotation of Transposable Elements and Repeats in Maize. 玉米转座元件和重复序列全基因组注释的四种策略

Cold Spring Harbor protocols Pub Date : 2025-09-02 DOI: 10.1101/pdb.prot108578

Christopher W Benson, Pedro Heringer, Shujun Ou

{"title":"Four Strategies for Whole-Genome Annotation of Transposable Elements and Repeats in Maize.","authors":"Christopher W Benson, Pedro Heringer, Shujun Ou","doi":"10.1101/pdb.prot108578","DOIUrl":"10.1101/pdb.prot108578","url":null,"abstract":"Transposable elements (TEs) and tandem repeat arrays are ubiquitous components of genomes across all domains of life. Many types of repetitive DNA do not appear to encode for functional proteins, and those that do, typically only code for enzymes involved in their own replication. Nevertheless, repetitive DNA sequences can significantly alter genome structure, and can have a profound impact on an organism's biology at both the molecular and organismal levels. Advances in long-read sequencing technology have enabled the resolution of previously collapsed contigs and scaffolds that are rich in repeats, which has made the accurate annotation of TEs and other repetitive sequences a crucial early step in genome analysis. Here, we provide a detailed tutorial for streamlined annotation of TEs and repeats in the genome of the model plant Zea mays (maize). Maize is ideally suited to illustrate these procedures due to its repeat-rich genome and the volume of publicly available and high-quality genomic resources. We outline four possible approaches for TE and repeat annotation, each aimed at accommodating a different set of scientific interests. Additionally, we demonstrate how to evaluate annotation quality, and provide scripts to help graphically depict TE and repeat landscapes. Although the protocol is tailored for maize, we also offer pointers for researchers working on other systems throughout and expect that these procedures will be broadly applicable to any eukaryotic genome.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108578"},"PeriodicalIF":0.0,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142139526","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

Root Anatomy: Preparing, Imaging, and Analyzing Maize Root Cross-Sections. 根部解剖：制备、成像和分析玉米根横截面。

Cold Spring Harbor protocols Pub Date : 2025-09-02 DOI: 10.1101/pdb.prot108585

Jagdeep Singh Sidhu, Hannah M Schneider

{"title":"Root Anatomy: Preparing, Imaging, and Analyzing Maize Root Cross-Sections.","authors":"Jagdeep Singh Sidhu, Hannah M Schneider","doi":"10.1101/pdb.prot108585","DOIUrl":"10.1101/pdb.prot108585","url":null,"abstract":"Root anatomy plays a critical structural and functional role in the maize root system, and regulates edaphic stress tolerance. The function and genetic basis of several maize root anatomical traits for stress tolerance have been demonstrated. Leveraging root anatomical traits in maize thus holds great potential for developing cultivars with greater nutrient and water efficiency. Key for such approaches is the ability to characterize the root anatomy of plants of interest. Here, we outline a systematic method for preparing, imaging, and analyzing maize root cross-sections. The protocol describes root sectioning (by hand or using a vibratome), preparation of microscope slides and toluidine blue staining, imaging under a light microscope, and both manual and semiautomated methods for anatomical feature extraction from images. The protocol enables the visualization and quantification of various anatomical tissues and traits, and its simplicity, adaptability, and accessibility make it an ideal choice for both small- and large-scale phenotyping studies in maize and other plant species. This standardized protocol provides researchers with a comprehensive methodology to accurately dissect root structures, enabling in-depth analyses that are essential for understanding plant growth, development, and adaptive value for stress tolerance.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108585"},"PeriodicalIF":0.0,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142105086","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

Classical Conditioning of Adult Drosophila. 成年果蝇的经典条件反射

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.prot108566

Zeynep Okray, Pedro F Jacob, John-Paul Moszynski, Clifford B Talbot, Scott Waddell

{"title":"Classical Conditioning of Adult Drosophila.","authors":"Zeynep Okray, Pedro F Jacob, John-Paul Moszynski, Clifford B Talbot, Scott Waddell","doi":"10.1101/pdb.prot108566","DOIUrl":"10.1101/pdb.prot108566","url":null,"abstract":"Olfactory classical conditioning paradigms have been extensively used since the early 1970s to apply genetic approaches to the study of memory in Drosophila. Over the intervening years, advances in genetics have largely changed the focus of studies from the role of single genes in memory to investigation of memory-relevant neuronal circuits. However, the original behavioral paradigms have remained largely unaltered, besides investigators making a few useful tweaks to the training and testing apparatus and modifications to the operating procedures. In this protocol, we provide the reader with a detailed description of the manufacture and assembly of a typical T-maze apparatus, where populations of adult flies can be trained and their odor memory tested later, by giving them a binary choice between the two trained odors. We describe how variations of the training apparatus permit both aversive (odor-shock) and appetitive (odor-sugar) memories to be studied. In addition, we describe a recent modification of the apparatus and protocol that permits study of multisensory (color and odor) aversive and appetitive learning. Control assays for sensory acuity and locomotion are also included.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108566"},"PeriodicalIF":0.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399629","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

Twists to Classical Conditioning of Adult Drosophila. 成年果蝇经典条件反射的转折

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.top108567

Zeynep Okray, Scott Waddell

{"title":"Twists to Classical Conditioning of Adult Drosophila.","authors":"Zeynep Okray, Scott Waddell","doi":"10.1101/pdb.top108567","DOIUrl":"10.1101/pdb.top108567","url":null,"abstract":"Memory has been extensively studied in Drosophila since the early 1970s. Straightforward aversive and appetitive conditioning paradigms train populations of flies to associate the pairing of one of two odors with either punishment or reward. After training, the flies show either preferential avoidance or approach behavior, to the appropriate odor, when given a choice between the two odors in a simple T-maze apparatus. These basic experimental approaches have proven useful in understanding the genetic, molecular, cellular, and neuronal network bases of various valence-specific memories in the fly brain. In addition, numerous modifications to these assays have permitted the study of a broad range of memory-related phenomena. Labile short-term avoidance and approach memories can be readily distinguished from more stable \"consolidated\" long-term memory equivalents. Prior or subsequent experience of the training cues, and manipulations of the flies' condition, have revealed how parallel competing memories and incompatible states can temporarily interfere with memory retrieval, providing insight into mechanisms of forgetting. Recent studies have also modified the training and testing apparatus to allow simultaneous presentation of odors and colors, providing insight into mechanisms of multisensory learning.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108567"},"PeriodicalIF":0.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399630","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 VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning. 制备 VCSM13 辅助噬菌体，用于显示文库重扩增和生物平移。

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.prot108569

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

引用次数: 0

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation. 鸡免疫后提取 RNA 和 cDNA 合成用于抗体库制备。

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.prot108568

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

{"title":"Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.","authors":"Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung","doi":"10.1101/pdb.prot108568","DOIUrl":"10.1101/pdb.prot108568","url":null,"abstract":"Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an \"immune library\" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108568"},"PeriodicalIF":0.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141995465","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

Generation of Chicken Antibody Libraries and Selection of Antigen Binders. 鸡抗体库的生成和抗原结合体的选择。

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.top108210

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

{"title":"Generation of Chicken Antibody Libraries and Selection of Antigen Binders.","authors":"Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung","doi":"10.1101/pdb.top108210","DOIUrl":"10.1101/pdb.top108210","url":null,"abstract":"Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108210"},"PeriodicalIF":0.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141995467","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

Selection of Antigen Binders from a Chicken Single-Chain Variable Fragment Library. 从鸡单链可变片段库中选择抗原结合剂

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.prot108211

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

引用次数: 0

Generation of a Phage Display Chicken Single-Chain Variable Fragment Library. 噬菌体展示鸡单链可变片段库的生成。

Cold Spring Harbor protocols Pub Date : 2025-08-01 DOI: 10.1101/pdb.prot108213

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

引用次数: 0