Current protocols in mouse biology最新文献_第2页

Current protocols in mouse biology Pub Date : 2020-06-04 DOI: 10.1002/cpmo.76

Jeremy Sanderson

{"title":"Fundamentals of Microscopy","authors":"Jeremy Sanderson","doi":"10.1002/cpmo.76","DOIUrl":"10.1002/cpmo.76","url":null,"abstract":"The light (or optical) microscope is the icon of science. The aphorism “seeing is believing” is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high-quality images. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Setting up Köhler illumination for a brightfield microscopeBasic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscopeBasic Protocol 3: Generic protocol for operating a confocal microscope","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.76","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38012636","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: 6

Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments 用于原位杂交实验的地高辛标记核糖探针的制备

Current protocols in mouse biology Pub Date : 2020-05-21 DOI: 10.1002/cpmo.74

Kristen S. Barratt, Ruth M. Arkell

引用次数: 6

Whole-Mount In Situ Hybridization in Post-Implantation Staged Mouse Embryos 植入后分期小鼠胚胎的全贴装原位杂交

Current protocols in mouse biology Pub Date : 2020-05-21 DOI: 10.1002/cpmo.75

Kristen S. Barratt, Ruth M. Arkell

{"title":"Whole-Mount In Situ Hybridization in Post-Implantation Staged Mouse Embryos","authors":"Kristen S. Barratt, Ruth M. Arkell","doi":"10.1002/cpmo.75","DOIUrl":"10.1002/cpmo.75","url":null,"abstract":"Understanding RNA expression in space and time is a key initial step in dissecting gene function. The ability to visualize gene expression in whole-tissue or whole-specimen preparations, called in situ hybridization (ISH), was first developed 50 years ago. Two decades later, these protocols were adapted to establish robust methods for whole-mount ISH to murine embryos. The precise protocols vary somewhat between early-gestation and mid-gestation mouse embryos; the protocol presented here is optimal for use with post-implantation stage mouse embryos (stages 5.5–9.5 dpc). Routine uses of whole-mount ISH include documenting the wild-type expression pattern of individual genes and comparison of the expression pattern of signature genes (i.e., those that identify particular cells and tissues within an embryo) between wild-type and mutant embryos as part of a phenotyping experiment. This technique remains a mainstay of developmental biology studies and complements the massively parallel assessment of gene expression from dissociated tissues and cells via RNA-sequencing techniques. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Dissection of post-implantation (5.5-9.5 dpc) murine embryosBasic Protocol 2: Whole-mount in situ hybridization in post-implantation embryosBasic Protocol 3: Visualization of post-WMISH embryosSupport Protocol 1: Creation of siliconized glass pipettesSupport Protocol 2: Creation of embryo powder","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.75","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37961837","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: 5

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents 用CRISPR-Cas9试剂生成和分型基因组编辑小鼠的简单方案

Current protocols in mouse biology Pub Date : 2020-03-11 DOI: 10.1002/cpmo.69

Almudena Fernández, Matías Morín, Diego Muñoz-Santos, Santiago Josa, Andrea Montero, Marcos Rubio-Fernández, Marta Cantero, Julia Fernández, María Jesús del Hierro, Marta Castrillo, Miguel Ángel Moreno-Pelayo, Lluís Montoliu

{"title":"Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents","authors":"Almudena Fernández, Matías Morín, Diego Muñoz-Santos, Santiago Josa, Andrea Montero, Marcos Rubio-Fernández, Marta Cantero, Julia Fernández, María Jesús del Hierro, Marta Castrillo, Miguel Ángel Moreno-Pelayo, Lluís Montoliu","doi":"10.1002/cpmo.69","DOIUrl":"10.1002/cpmo.69","url":null,"abstract":"The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategyBasic Protocol 2: Preparing and validating CRISPR-Cas9 reagentsBasic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytesBasic Protocol 4: Genotyping genome-edited miceSupport Protocol: Genotyping for CRISPR-generated “indel” mutations","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37726603","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: 10

A Novel One-Day Learning Procedure for Mice 一种新的小鼠一天学习程序

Current protocols in mouse biology Pub Date : 2020-02-25 DOI: 10.1002/cpmo.68

Martin Darvas, Kishore Mukherjee, Amanda Lee, Warren Ladiges

引用次数: 13

Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR 利用2细胞(2C)-同源重组(HR)-CRISPR高效生成大片段敲入小鼠模型

Current protocols in mouse biology Pub Date : 2020-01-08 DOI: 10.1002/cpmo.67

Bin Gu, Eszter Posfai, Marina Gertsenstein, Janet Rossant

{"title":"Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR","authors":"Bin Gu, Eszter Posfai, Marina Gertsenstein, Janet Rossant","doi":"10.1002/cpmo.67","DOIUrl":"10.1002/cpmo.67","url":null,"abstract":"Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9–monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Single guide RNA and repair template designBasic Protocol 2: Preparing reagents for 2C-HR-CRISPRBasic Protocol 3: Microinjecting 2-cell-stage mouse embryos","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.67","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37521314","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: 26

Issue Information TOC 发布信息TOC

Current protocols in mouse biology Pub Date : 2019-12-19 DOI: 10.1002/cpmo.55

引用次数: 0

Designing a QTL Mapping Study for Implementation in the Realized Collaborative Cross Genetic Reference Population 设计一种QTL定位研究，并在已实现的协同交叉遗传参考群体中实现

Current protocols in mouse biology Pub Date : 2019-10-16 DOI: 10.1002/cpmo.66

Morris Soller, Hanifa J. Abu-Toamih Atamni, Ilona Binenbaum, Aristotelis Chatziioannou, Fuad A. Iraqi

{"title":"Designing a QTL Mapping Study for Implementation in the Realized Collaborative Cross Genetic Reference Population","authors":"Morris Soller, Hanifa J. Abu-Toamih Atamni, Ilona Binenbaum, Aristotelis Chatziioannou, Fuad A. Iraqi","doi":"10.1002/cpmo.66","DOIUrl":"10.1002/cpmo.66","url":null,"abstract":"The Collaborative Cross (CC) mouse resource is a next-generation mouse genetic reference population (GRP) designed for high-resolution mapping of quantitative trait loci (QTL) of large effect affecting complex traits during health and disease. The CC resource consists of a set of 72 recombinant inbred lines (RILs) generated by reciprocal crossing of five classical and three wild-derived mouse founder strains. Complex traits are controlled by variations within multiple genes and environmental factors, and their mutual interactions. These traits are observed at multiple levels of the animals’ systems, including metabolism, body weight, immune profile, and susceptibility or resistance to the development and progress of infectious or chronic diseases. Herein, we present general guidelines for design of QTL mapping experiments using the CC resource—along with full step-by-step protocols and methods that were implemented in our lab for the phenotypic and genotypic characterization of the different CC lines—for mapping the genes underlying host response to infectious and chronic diseases. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: CC lines for whole body mass index (BMI)Basic Protocol 2: A detailed assessment of the power to detect effect sizes based on the number of lines used, and the number of replicates per lineBasic Protocol 3: Obtaining power for QTL with given target effect by interpolating in Table 1 of Keele et al. (2019)","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.66","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48338772","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: 5

Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation 优化小鼠基因分型PCR:可靠、快速、经济、稳健和适用于任何类型突变的高通量基因分型方案的建议

Current protocols in mouse biology Pub Date : 2019-09-26 DOI: 10.1002/cpmo.65

Sylvie Jacquot, Nathalie Chartoire, Françoise Piguet, Yann Hérault, Guillaume Pavlovic

{"title":"Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation","authors":"Sylvie Jacquot, Nathalie Chartoire, Françoise Piguet, Yann Hérault, Guillaume Pavlovic","doi":"10.1002/cpmo.65","DOIUrl":"10.1002/cpmo.65","url":null,"abstract":"Genotyping consists of searching for a DNA sequence variation localized at a well-defined locus in the genome. It is an essential step in animal research because it allows the identification of animals that will be bred to generate and maintain a colony, euthanized to control the available space in the animal facility, or used in experiment protocols. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost-effective characterization of mouse genotype. We discuss optimization of parameters to improve the reliability of each assay and propose recommendations for enhancing reproducibility and reducing the occurrence of inconclusive genotyping. All steps required for efficient genotyping are presented: tissue collection; sample verification and direct DNA lysis; establishment of a robust genotyping strategy with reliable, rapid, and cost-effective assays; and finally, transition to high-throughput automatized PCR, including mix miniaturization and automation. © 2019 The Authors.Basic Protocol 1: Tissue sampling methods and procedureBasic Protocol 2: Sample verification and DNA lysisBasic Protocol 3: Design of a genotyping strategyBasic Protocol 4: Moving to high-throughput genotyping","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.65","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44818259","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: 13

Issue Information TOC 发布信息TOC

Current protocols in mouse biology Pub Date : 2019-09-18 DOI: 10.1002/cpmo.54

引用次数: 0