Current protocols最新文献_第8页

Working with Miraculous Mice: Mus musculus as a Model Organism 与神奇的小鼠共事：作为模式生物的麝。

Current protocols Pub Date : 2024-10-22 DOI: 10.1002/cpz1.70021

Anick Standley, Jinhan Xie, Angelica WY Lau, Lauren Grote, Andrew J. Gifford

{"title":"Working with Miraculous Mice: Mus musculus as a Model Organism","authors":"Anick Standley, Jinhan Xie, Angelica WY Lau, Lauren Grote, Andrew J. Gifford","doi":"10.1002/cpz1.70021","DOIUrl":"10.1002/cpz1.70021","url":null,"abstract":"The laboratory mouse has been described as a “miracle” model organism, providing a window by which we may gain an understanding of ourselves. Since the first recorded mouse experiment in 1664, the mouse has become the most used animal model in biomedical research. Mice are ideally suited as a model organism because of their small size, short gestation period, large litter size, and genetic similarity to humans. This article provides a broad overview of the laboratory mouse as a model organism and is intended for undergraduates and those new to working with mice. We delve into the history of the laboratory mouse and outline important terminology to accurately describe research mice. The types of laboratory mice available to researchers are reviewed, including outbred stocks, inbred strains, immunocompromised mice, and genetically engineered mice. The critical role mice have played in advancing knowledge in the areas of oncology, immunology, and pharmacology is highlighted by examining the significant contribution of mice to Nobel Prize winning research. International mouse mutagenesis programs and accurate phenotyping of mouse models are outlined. We also explain important considerations for working with mice, including animal ethics; the welfare principles of replacement, refinement, and reduction; and the choice of mouse model in experimental design. Finally, we present practical advice for maintaining a mouse colony, which involves adequate training of staff, the logistics of mouse housing, monitoring colony health, and breeding strategies. Useful resources for working with mice are also listed. The aim of this overview is to equip the reader with a broad appreciation of the enormous potential and some of the complexities of working with the laboratory mouse in a quest to improve human health. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70021","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142483998","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

Generation of Murine Cancer Cell Lines 生成小鼠癌症细胞系。

Current protocols Pub Date : 2024-10-21 DOI: 10.1002/cpz1.70037

Cheryl Zhang, Anthony Lin, Xiang Chen, Yi-Chieh Nancy Du

引用次数: 0

Using Spectral Flow Cytometry to Characterize Anti-Tumor Immunity in Orthotopic and Subcutaneous Mouse Models of Cancer 利用光谱流式细胞术鉴定正位和皮下小鼠癌症模型的抗肿瘤免疫特性

Current protocols Pub Date : 2024-10-21 DOI: 10.1002/cpz1.70032

Giampiero Valenzano, Shannon N. Russell, Simei Go, Eric O'Neill, Keaton I. Jones

{"title":"Using Spectral Flow Cytometry to Characterize Anti-Tumor Immunity in Orthotopic and Subcutaneous Mouse Models of Cancer","authors":"Giampiero Valenzano, Shannon N. Russell, Simei Go, Eric O'Neill, Keaton I. Jones","doi":"10.1002/cpz1.70032","DOIUrl":"10.1002/cpz1.70032","url":null,"abstract":"Mouse models remain at the forefront of immuno-oncology research, providing invaluable insights into the complex interactions between the immune system and developing tumors. While several flow cytometry panels have been developed to study cancer immunity in mice, most are limited in their capacity to address the complexity of anti-cancer immune responses. For example, many of the panels developed to date focus on a restricted number of leukocyte populations (T cells or antigen-presenting cells), failing to include the multitude of other subsets that participate in anti-cancer immunity. In addition, these panels were developed using blood or splenic leukocytes. While the immune composition of the blood or spleen can provide information on systemic immune responses to cancer, it is in the tumor microenvironment (TME) that local immunity takes place. Therefore, we optimized this spectral flow cytometry panel to identify the chief cell types that take part in cancer immunity using immune cells from cancer tissue. We used pancreatic tumors implanted both orthotopically and subcutaneously to demonstrate the panel's flexibility and suitability in diverse mouse models. The panel was also validated in peripheral immune districts (the blood, spleen, and liver of tumor-bearing mice) to allow comparisons between local and systemic anti-tumor immunity. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Tumor induction—OrthotopicAlternate Protocol: Tumor induction—SubcutaneousBasic Protocol 2: Preparation of single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing miceBasic Protocol 3: Staining single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70032","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142483997","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

De Novo Synthesis of Error-Free Long Oligos 从头合成无差错长寡糖

Current protocols Pub Date : 2024-10-18 DOI: 10.1002/cpz1.70028

Shiyue Fang, Reed Arneson, Yipeng Yin, Yinan Yuan

{"title":"De Novo Synthesis of Error-Free Long Oligos","authors":"Shiyue Fang, Reed Arneson, Yipeng Yin, Yinan Yuan","doi":"10.1002/cpz1.70028","DOIUrl":"https://doi.org/10.1002/cpz1.70028","url":null,"abstract":"This protocol describes the synthesis of long oligonucleotides (up to 401-mer), their isolation from complex mixtures using the catching-by-polymerization (CBP) method, and the selection of error-free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid-phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full-length sequence with a polymerizable tagging phosphoramidite (PTP), co-polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full-length sequence from the polymer. Cloning and sequencing guided selection of error-free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher-order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Long oligo synthesisSupport Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP)Support Protocol 2: Synthesis of 5′-O-Bz phosphoramiditeBasic Protocol 2: Catching-by-polymerization (CBP) purificationBasic Protocol 3: Error-free sequence selection via cloning and sequencing","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142451242","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

Behavioral Assays for Comprehensive Evaluation of Cognitive and Neuropsychiatric Comorbidities of Traumatic Brain Injury and Chronic Neurological Disorders 用于全面评估创伤性脑损伤和慢性神经系统疾病的认知和神经精神并发症的行为测定法

Current protocols Pub Date : 2024-10-18 DOI: 10.1002/cpz1.70019

Doodipala Samba Reddy, Yue Li, Taha Qamari, Sreevidhya Ramakrishnan

{"title":"Behavioral Assays for Comprehensive Evaluation of Cognitive and Neuropsychiatric Comorbidities of Traumatic Brain Injury and Chronic Neurological Disorders","authors":"Doodipala Samba Reddy, Yue Li, Taha Qamari, Sreevidhya Ramakrishnan","doi":"10.1002/cpz1.70019","DOIUrl":"https://doi.org/10.1002/cpz1.70019","url":null,"abstract":"Neurological deficits, psychiatric disorders, and cognitive impairments often accompany stroke, brain injury, epilepsy, and many neurological disorders, which present intricate comorbidities that challenge recognition and management. There are many tools and paradigms for evaluating learning, memory, anxiety, and depression-like behaviors in lab animal models of brain disorders. However, there is a significant gap between clinical observations and experimental models, which limit understanding of the complex interplay between chronic brain conditions and their impact on cognitive dysfunction and psychiatric impairments. This article describes an overview of experimental rationale, methods, protocols, and strategies for evaluating sensorimotor, affective and cognitive-associated comorbid behaviors in epilepsy, traumatic brain injury (TBI), stroke, spinal cord injury (SCI), and many other neurological disorders. First, we delve into clinical evidence elucidating the profound impact of comorbidities, e.g., psychiatric disorders and cognitive deficits, in individuals with epilepsy. Then, we discuss diverse approaches to assess these comorbidities in experimental models of brain diseases. Finally, we explore the methodologies for assessing motor function, sensorimotor, behavior, and psychiatric health. We cover strategies and protocols enabling these assays, including implementing behavioral paradigms to assess learning and memory, anxiety, and depression-like behaviors in rodents in health and disease conditions. It is essential to consider a comprehensive battery of tests to investigate various behavioral deficits, considering environment, age, and sex differences relevant to the disease, such as TBI, SCI, epilepsy, stroke, and other complex neurological conditions. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70019","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142449203","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

Evaluation of Cardiotoxicity of Cancer Chemotherapeutics Using Daphnia magna as a Preclinical Model 以大型蚤为临床前模型评估癌症化疗药物的心脏毒性

Current protocols Pub Date : 2024-10-17 DOI: 10.1002/cpz1.70035

Qudrathulla Khan Quadri Mohammed, Rama Krishna Kancha

{"title":"Evaluation of Cardiotoxicity of Cancer Chemotherapeutics Using Daphnia magna as a Preclinical Model","authors":"Qudrathulla Khan Quadri Mohammed, Rama Krishna Kancha","doi":"10.1002/cpz1.70035","DOIUrl":"https://doi.org/10.1002/cpz1.70035","url":null,"abstract":"One of the major concerns following cancer treatment is cardiotoxicity. Therefore, it is important to predict potential cardiotoxicity of cancer chemotherapeutics at the preclinical phase. Current models of cardiotoxicity testing involve either cell culture models or rodent models. We developed a simple invertebrate animal model for rapid screening of cardiotoxicity of cancer chemotherapeutics. Daphnia magna (water flea, a crustacean) has a transparent body and a large myogenic heart that can be easily monitored under a microscope. Using this model, we have previously described comparative cardiotoxicity of several kinase inhibitors that were approved for the treatment of multiple cancers. In this article, we describe the step-wise protocols for evaluating the heart rate and survival of D. magna with relevant information on troubleshooting. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Culturing and maintenance of D. magnaBasic Protocol 2: Experimental design for evaluating heart rate of DaphniaBasic Protocol 3: Long-term effect on Daphnia survival upon drug exposure","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142449212","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

Correction: Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells 更正：从多能干细胞中生成人类腹侧中脑有组织细胞

Current protocols Pub Date : 2024-10-16 DOI: 10.1002/cpz1.70042

Edoardo Sozzi, Fredrik Nilsson, Janko Kajtez, Malin Parmar, Alessandro Fiorenzano

引用次数: 0

Genetic Modification of Mice Using Prime Editing 利用基因编辑技术修改小鼠基因

Current protocols Pub Date : 2024-10-15 DOI: 10.1002/cpz1.70034

Amr R. Salem, Xiaoling Xie, Susan H. Griffin, Lin Gan, Joseph M Miano

{"title":"Genetic Modification of Mice Using Prime Editing","authors":"Amr R. Salem, Xiaoling Xie, Susan H. Griffin, Lin Gan, Joseph M Miano","doi":"10.1002/cpz1.70034","DOIUrl":"https://doi.org/10.1002/cpz1.70034","url":null,"abstract":"Genetically modifying mice traditionally involved complex methods of designing and validating targeting constructs, embryonic stem cell electroporation and selection, blastocyst injection, and breeding chimeras for germline transmission. Such arduous steps were best carried out by specialized gene targeting cores in academia or through expensive commercial vendors. Further, the time from initiation to completion of a project often took at least 1 year and, in some cases, much longer (or never), with no guarantees of success. The RNA-programmable CRISPR system of gene editing has greatly streamlined the generation of gene modifications (e.g., small substitutions, insertions, and deletions) in the mouse with high rates of success. Several editing platforms exist for gene/genome targeting in mice and other animal models previously difficult or impossible to alter. Here, we provide a simplified method of generating genetically modified mice using the prime editing platform. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Design, cloning, and synthesis of engineered pegRNA (epegRNA)Basic Protocol 2: Microinjection of PE2 components into mouse zygoteBasic Protocol 3: Genotyping founder mice and breeding for germline transmission","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142438984","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

A Practical Guide to 3D Printing for Chemistry and Biology Laboratories 化学和生物实验室 3D 打印实用指南

Current protocols Pub Date : 2024-10-15 DOI: 10.1002/cpz1.70036

Arjun S. Pamidi, Michael B. Spano, Gregory A. Weiss

{"title":"A Practical Guide to 3D Printing for Chemistry and Biology Laboratories","authors":"Arjun S. Pamidi, Michael B. Spano, Gregory A. Weiss","doi":"10.1002/cpz1.70036","DOIUrl":"https://doi.org/10.1002/cpz1.70036","url":null,"abstract":"Three-dimensional (3D) printing promises a revolution in laboratory creativity by enabling rapid prototyping, broader availability of scientific apparatuses, and transformative scientific workflows. We believe all chemistry and biology laboratories should equip themselves with one or more 3D printers and a critical mass of scientists trained to operate them. This overview surveys the techniques, intricacies, and pitfalls associated with 3D printing of functional parts, including measurements, computer-aided design, slicing, limitations of 3D printing, troubleshooting, tips for tricky filaments, and 3D printer maintenance. A flow cells are essential tools in chemistry and biology laboratories, we discuss techniques relevant to the construction of watertight 3D-printed parts. Finally, we articulate a set of principles required for reporting 3D-printed innovations to improve the field's reproducibility and encourage iterative improvements by other scientists. Ideally, authors, peer reviewers, and editors will adopt these principles. We hope these protocols inspire a new generation of publications applying 3D printing in chemistry and biology—especially highly reproducible inventions with the requisite detail and associated documentation. Such reports will facilitate broad adoption and creative iteration of the most innovative designs, thus accelerating discovery in chemistry and biology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70036","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142438985","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

Using Reporter Gene Assays to Screen and Identify Chemical Compounds that Modulate Estrogen Receptor Activity 利用报告基因测定筛选和鉴定可调节雌激素受体活性的化学化合物

Current protocols Pub Date : 2024-10-15 DOI: 10.1002/cpz1.70029

Masato Ooka, Srilatha Sakamuru, J. David Furlow, Menghang Xia

{"title":"Using Reporter Gene Assays to Screen and Identify Chemical Compounds that Modulate Estrogen Receptor Activity","authors":"Masato Ooka, Srilatha Sakamuru, J. David Furlow, Menghang Xia","doi":"10.1002/cpz1.70029","DOIUrl":"https://doi.org/10.1002/cpz1.70029","url":null,"abstract":"Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor αBasic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70029","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142438986","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