Cold Spring Harbor protocols最新文献_第3页

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

A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards. 蜥蜴卵母细胞注射手术方法及CRISPR-Cas9诱变。

Cold Spring Harbor protocols Pub Date : 2025-07-31 DOI: 10.1101/pdb.prot108652

Christina E Sabin, Sukhada P Samudra, Anna L Iouchmanov, Amber L Rittgers, James D Lauderdale, Douglas B Menke

{"title":"A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards.","authors":"Christina E Sabin, Sukhada P Samudra, Anna L Iouchmanov, Amber L Rittgers, James D Lauderdale, Douglas B Menke","doi":"10.1101/pdb.prot108652","DOIUrl":"10.1101/pdb.prot108652","url":null,"abstract":"Squamates, the taxon that comprises lizards and snakes, are a diverse assemblage of reptiles represented by more than 11,000 described species. Studies of gene function in squamates, however, have remained very limited, largely due to the lack of established genetic tools and suitable experimental systems. A major challenge for the development of CRISPR-based gene editing in these reptiles is that the isolation of fertilized oocytes or single-celled embryos is impractical for most species, given that fertilization occurs internally, the females of many species can store sperm, and simple methods for detecting ovulation are lacking. To overcome these challenges, we have developed a unique surgical approach in the brown anole lizard Anolis sagrei The procedure enables users to access and microinject unfertilized oocytes while they are still maturing within the lizard ovary. We describe here the methods to anesthetize adult female anoles, access the ovary through a surgical incision into the coelomic cavity, and microinject unfertilized oocytes with CRISPR-Cas9 ribonucleoprotein complexes to generate targeted mutations, enabling the routine production of gene-edited lizards.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12376173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144759366","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

Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution. 蜥蜴作为爬行动物发育和进化中基因功能研究的模式系统。

Cold Spring Harbor protocols Pub Date : 2025-07-31 DOI: 10.1101/pdb.top108535

Christina E Sabin, James D Lauderdale, Douglas B Menke

{"title":"Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution.","authors":"Christina E Sabin, James D Lauderdale, Douglas B Menke","doi":"10.1101/pdb.top108535","DOIUrl":"10.1101/pdb.top108535","url":null,"abstract":"Anolis lizards are an ecologically diverse group that includes more than 400 described species. These reptiles have been the subject of wide-ranging studies, from speciation and convergent evolution to climate adaptation and tail regeneration. While CRISPR-based gene editing has tremendous potential to reveal new insights into these and other aspects of Anolis biology, the reproductive biology of these reptiles has presented significant barriers to gene editing. Here, we briefly summarize gene editing approaches in vertebrates and discuss some of the major challenges associated with the performance of gene editing in anoles. We then introduce a recently established surgical procedure that enables the injection of CRISPR-Cas into the developing oocytes of female lizards. This approach circumvents the need to manipulate early-stage embryos and permits the production of gene-edited anoles. This method has recently been successfully adapted for use in other reptiles, suggesting that it may be effective in a wide range of species and will broadly enable studies of gene function in reptiles.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12376193/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144759365","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

Inoculation of Maize Roots with Globisporangium ultimum var. ultimum to Study Pythium Root Rot. 用最后一种球孢根接种玉米根腐病研究。

Cold Spring Harbor protocols Pub Date : 2025-07-16 DOI: 10.1101/pdb.prot108640

Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros

{"title":"Inoculation of Maize Roots with Globisporangium ultimum var. ultimum to Study Pythium Root Rot.","authors":"Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros","doi":"10.1101/pdb.prot108640","DOIUrl":"10.1101/pdb.prot108640","url":null,"abstract":"Maize is a globally important field crop for food and fuel production. Yield can be affected early in the growing season by oomycete and fungal pathogens that cause root rot or prevent seed germination. The diseases caused by these pathogens are referred to as seedling blights, root rots, or damping off. Pythium root rot is one of the most significant of these diseases. The disease is caused by multiple species of the oomycete genera Globisporangium and Pythium and results in significant yield losses due to reduced seed germination and reduced vigor of surviving seedlings. In this protocol, we mimic the natural infection process by mixing the inoculum into the potting media in which seeds are planted. Then, we flood the seeds daily for several days in large plastic totes to induce flooding conditions. Disease severity is assessed using stand counts and measuring root mass and length. This protocol allows researchers to investigate quantitative differences in disease symptoms, isolate aggressiveness, as well as levels of host resistance. This protocol was developed for the pathogen Globisporangium ultimum var. ultimum, but it can be adapted for other species.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215157","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

Corrigendum: Inoculation of Maize Roots with Globisporangium ultimum var. ultimum to Study Pythium Root Rot. 勘误：用最后一种球孢菊接种玉米根系研究牙周病。

Cold Spring Harbor protocols Pub Date : 2025-07-16 DOI: 10.1101/pdb.Corr108654

Harrison Hall, Peyton Sorensen, Tiffany Jamann, Santiago Mideros

引用次数: 0

The Drosophila Larval Neuromuscular Junction: Developmental Overview. 果蝇幼虫神经肌肉接头：发育概述

Cold Spring Harbor protocols Pub Date : 2025-07-01 DOI: 10.1101/pdb.top108449

James Ashley, Robert A Carrillo

引用次数: 0

Immunohistochemistry and Morphometric Analysis of Drosophila Larval Body Wall Neuromuscular Junction Preparations. 果蝇幼虫体壁神经肌肉接头制剂的免疫组织化学和形态计量分析

Cold Spring Harbor protocols Pub Date : 2025-07-01 DOI: 10.1101/pdb.prot108500

James Ashley, Robert A Carrillo

引用次数: 0

Using the Proximity Ligation Assay to Visualize Colocalization of Proteins at the Drosophila Larval Neuromuscular Junction. 利用近接测定法观察果蝇幼虫神经肌肉接头处蛋白质的共定位现象

Cold Spring Harbor protocols Pub Date : 2025-07-01 DOI: 10.1101/pdb.prot108502

James Ashley, Robert A Carrillo

{"title":"Using the Proximity Ligation Assay to Visualize Colocalization of Proteins at the Drosophila Larval Neuromuscular Junction.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108502","DOIUrl":"10.1101/pdb.prot108502","url":null,"abstract":"In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in the Drosophila NMJ.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108502"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310238","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

Drosophila Late Embryonic through Late Larval Stage Body Wall Dissection: Dissection Tools and Techniques. 果蝇胚后期至幼虫后期体壁解剖：解剖工具和技术。

Cold Spring Harbor protocols Pub Date : 2025-07-01 DOI: 10.1101/pdb.prot108499

James Ashley, Robert A Carrillo

{"title":"Drosophila Late Embryonic through Late Larval Stage Body Wall Dissection: Dissection Tools and Techniques.","authors":"James Ashley, Robert A Carrillo","doi":"10.1101/pdb.prot108499","DOIUrl":"10.1101/pdb.prot108499","url":null,"abstract":"One of the challenges of studying synaptic structure and function is accessibility. Some of the earliest readily identifiable and accessible synapses were from the frog and various arthropods. To address questions regarding mechanisms that underlie synaptic development and function, genetically tractable systems were required, and researchers turned to the Drosophila melanogaster embryonic/larval neuromuscular preparation. Drosophila embryos are transparent and can be labeled with antibodies or probes and imaged in whole-mount preparation for structural analysis. Embryos can also be dissected to visualize the entire body wall musculature as well as finer details including live protein trafficking and protein-protein interactions. Whereas younger dissected embryos can be mounted directly onto charged slides, more mature embryos and larvae develop a cuticle that impedes this adherence, so different techniques must be applied. In this protocol, we detail how to manufacture dissection tools and collect embryos, and discuss the individual steps of dissecting late-stage embryos, early first-instar larvae, and late-stage third-instar larvae.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108499"},"PeriodicalIF":0.0,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310233","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