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This detailed volume provides thorough protocols describing how to use genetics to study mouse and zebrafish erythropoiesis in whole animal models and for genetically manipulating cultured mouse and human erythroid cells. Protocols include strategies to analyze circulating red blood cell parameters, as well as progenitor cell status, stage of differentiation, and enucleation. Methods for the study of erythroid cell gene regulation using chromatin immunoprecipitation and chromatin conformation capture are detailed. Furthermore, protocols are provided to examine stress erythropoiesis, erythroblastic islands, and erythroblast structure. The collection also includes reviews on the available mouse models of erythropoiesis, as well as on future considerations for good manufacturing practice in order to translate the manufacture of erythrocytes to the clinic. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Erythropoiesis: Methods and Protocols provides a definitive source for molecular and cellular biology protocols to study erythropoiesis and to move this vital field forward.
List of contents
An Introduction to Erythropoiesis Approaches.- Using the Zebrafish as an Approach to Examine the Mechanisms of Vertebrate Erythropoiesis.- Mouse Models of Erythropoiesis and Associated Diseases.- Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells.- Stress Erythropoiesis Model Systems.- Approaches for Analysis of Erythroid Cell Parameters and Hemoglobinopathies in Mouse Models.- Functional Analysis of Erythroid Progenitors by Colony-Forming Assays.- Analyzing the Formation, Morphology, and Integrity of Erythroblastic Islands.- Flow Cytometry (FCM) Analysis and Fluorescence-Activated Cell Sorting (FACS) of Erythroid Cells.- Analysis of Erythropoiesis Using Imaging Flow Cytometry.- Flow Cytometric Analysis of Erythroblast Enucleation.- High-Resolution Fluorescence Microscope Imaging of Erythroblast Structure.- Chromatin Immunoprecipitation (ChIP) with Erythroid Samples.- Chromosome Conformation Capture (3C and Higher) with Erythroid Samples.- Genome Editing of Erythroid Cell Culture Model Systems.- In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood.- Growing and Genetically Manipulating Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP) Cell Lines.- Good Manufacturing Practice (GMP) Translation of Advanced Cellular Therapeutics: Lessons for the Manufacture of Erythrocytes as Medicinal Products.
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Summary
This detailed volume provides thorough protocols describing how to use genetics to study mouse and zebrafish erythropoiesis in whole animal models and for genetically manipulating cultured mouse and human erythroid cells. Protocols include strategies to analyze circulating red blood cell parameters, as well as progenitor cell status, stage of differentiation, and enucleation. Methods for the study of erythroid cell gene regulation using chromatin immunoprecipitation and chromatin conformation capture are detailed. Furthermore, protocols are provided to examine stress erythropoiesis, erythroblastic islands, and erythroblast structure. The collection also includes reviews on the available mouse models of erythropoiesis, as well as on future considerations for good manufacturing practice in order to translate the manufacture of erythrocytes to the clinic. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Erythropoiesis: Methods and Protocols provides a definitive source for molecular and cellular biology protocols to study erythropoiesis and to move this vital field forward.
