Germline Stem Cells (Methods in Molecular Biology, 1463)

✍ Scribed by Michael Buszczak (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 232
• Category: Library

✦ Synopsis

This second edition provides updated and new chapters on selected genetic, molecular, biochemical, and cell biological techniques. Chapter’s guide readers through methods and principles on primordial germ cells and germline stem cells, however many of these principles can be applied to different types of adult stem cells. Written in the highly successful Methods in Molecular Biology series format, 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 cutting-edge, Germline Stem Cells: Second Edition aims to present the new findings and techniques that have developed since the first edition.

✦ Table of Contents

Preface
Contents
Contributors
1: Analysis of the C. elegans Germline Stem Cell Pool
1 Introduction
2 Materials
2.1 Reagents
2.2 Web Resources
3 Methods
3.1 Identification of the Progenitor Zone in Wild-Type C. elegans Hermaphrodites
3.2 Mounting Live Animals for Light Microscopy (see Notes 2 and 3)
3.3 Immuno-histochemistry of Extruded Gonads
3.4 Visualization of DNA and Germline Apoptosis in Live Animals [55, 62]
3.5 Scoring the Length and Cell Number in the Progenitor Zone
3.6 Characterization of the GSC Pool and GSC Number
3.7 Markers for Progenitor Zone and Early Stages of Differentiation
3.8 Markers for the Somatic Gonad
3.9 Cell Cycle Analysis
3.10 Mitotic Index with Anti-PH3
3.11 EdU Labeling
3.12 Label with EdU by Feeding
3.13 Label with EdU by Soaking
3.14 Calculating Labeling Index and Identifying G1 and G2 Cells
3.15 Estimating Total Length of the Cell Cycle
3.16 Measure Cell Cycle Length by Measuring G2
3.17 Measure Cell Cycle Length by Measuring G2+M+G1
3.18 Identification of Premeiotic Cells in the Progenitor Zone
3.19 Characterization of Germline Defects in Mutants
4 Notes
References
Chapter 2: Methods for Studying the Germline of the Human Parasite Schistosoma mansoni
1 Introduction
2 Materials
2.1 Parasite Separation, Fixation, and Permeabilization Components
2.2 EdU Labeling and Detection Components
2.3 Riboprobe Synthesis Components
2.4 In Situ Hybridization Components
2.5 Components for dsRNA Synthesis
3 Methods
3.1 Fixation and Permeabilization of Parasites for In Situ Hybridization and EdU Detection
3.2 In Vivo EdU Labeling in S. mansoni
3.3 Riboprobe Synthesis
3.4 Whole-Mount In Situ Hybridization
3.5 Generation of dsRNA for RNAi
3.6 RNAi in Adult S. mansoni
4 Notes
References
Chapter 3: Evaluation of the Asymmetric Division of Drosophila Male Germline Stem Cells
1 Introduction
2 Materials
2.1 Preparation of Sample
2.2 Useful Antibodies
3 Methods
3.1 Fly Culture
3.2 Whole-Mount Immuno-histochemistry of Drosophila Testes
3.3 Scoring Centrosome Orientation
3.4 Scoring Spindle Orientation
3.5 Evaluating the Centrosome Orientation Checkpoint
3.6 Generation of Heat-Shock-Induced Germline Clones
3.7 Assessment of Asymmetric vs. Symmetric Stem Cell Division
3.8 Microscopy of Living Tissue
4 Notes
References
Chapter 4: Live Imaging of the Drosophila Testis Stem Cell Niche
1 Introduction
2 Materials
2.1 Dissection of Testes from Adult Male Drosophila
2.2 Live Imaging of Drosophila Testes
2.3 Immunostaining Testes After Live Imaging
3 Methods
3.1 Preparing Imaging Dish
3.2 Dissecting and Mounting Whole Testes
3.3 Overnight Time-Lapse Imaging of the Testis Niche
3.4 Immuno-localization After Time-Lapse Imaging
4 Notes
References
Chapter 5: RNA Isolation from Early Drosophila Larval Ovaries
1 Introduction
2 Materials
2.1 Ovary Dissection
2.2 RNA Purification Reagents
3 Methods
3.1 Dissection of Larvae
3.2 RNA Purification
3.3 Assessing RNA Yield and Quality
4 Notes
References
Chapter 6: Live-Cell Imaging of the Adult Drosophila Ovary Using Confocal Microscopy
1 Introduction
2 Materials
2.1 Drosophila-Rearing Components
2.2 Drosophila Ovary-Dissecting Components
2.3 Media Components
2.4 Mounting and Microscopy
3 Methods
3.1 Preparing Flies
3.2 Preparing Ovaries
3.3 Mounting Ovarioles
3.4 Imaging
4 Notes
References
Chapter 7: Measurement of mRNA Poly(A) Tail Lengths in Drosophila Female Germ Cells and Germ-Line Stem Cells
1 Introduction
2 Materials
2.1 GSC and Cystoblast Generation and Dissection
2.2 Total RNA Extraction and DNase Digestion
2.3 PAT and ePAT Assays
2.4 PAT Visualization
3 Methods
3.1 Generation of GSCs, Cystoblasts, and Early Germ Cells
3.2 Dissection, RNA Extraction and DNase Digestion
3.3 Phenol-Chloroform Extraction and Isopropanol Precipitation
3.4 LM-PAT cDNA Synthesis
3.5 ePAT cDNA Synthesis
3.6 PCR
4 Notes
References
Chapter 8: Identification of Germ-Line Stem Cells in Zebrafish
1 Introduction
2 Materials
2.1 Tissue Fixation and Permeabilization
2.2 Probe Synthesis
2.3 nanos2 In Situ Hybridization
2.4 Vasa Immunofluorescence
2.5 Mounting and Confocal Microscopy
3 Methods
3.1 Preparation of Template DNA
3.2 RNA Probe Synthesis
3.3 Preparation of Gonads for In Situ Hybridization and Indirect Immunostaining
3.4 Whole-Mount In Situ Hybridization
3.5 Antibody Staining
3.6 Mounting Ovaries for Confocal Analysis
4 Notes
References
Chapter 9: Primordial Germ Cell Isolation from Xenopus laevis Embryos
1 Introduction
2 Materials
2.1 Isolating PGCs from St. 12-16 X. laevis Embryos
2.2 Isolating PGCs from St. 24-42 X. laevis Embryos
3 Methods
3.1 Isolating PGCs from St. 12-16 X. laevis Embryos
3.2 Isolating PGCs from St. 24-42 X. laevis Embryos
4 Notes
References
10: Single-Cell Lineage Analysis of Oogenesis in Mice
1 Introduction
2 Materials
2.1 Mouse Housing and Breeding
2.2 Lineage Label Induction
2.3 Whole-Mount Immunostaining
2.4 Frozen Section Immunostaining
3 Methods
3.1 Mouse Crossing for Lineage Labeling
3.2 Tamoxifen Stock Preparation
3.3 Tmx Injection and Lineage Analysis of Single PGC in the Fetal Ovary
3.4 Collecting Fetal Ovaries
3.5 Whole-Mount Immunostaining of Fetal Ovaries
3.6 Tmx Injection for Lineage Analysis of GSCs in Adult Mouse Ovaries
3.7 Collecting Adult Mouse Ovaries
3.8 Frozen Sectioning Adult Mouse Ovaries
3.9 Immunohistological Staining of Ovarian Frozen Sections
4 Notes
References
11: Visualization and Lineage Tracing of Pax7+ Spermatogonial Stem Cells in the Mouse
1 Introduction
2 Materials
2.1 Immuno-histochemistry (IHC)
2.2 Immunofluoresence (IF)
2.3 Whole-Mount IF
3 Methods
3.1 IHC on Paraffin-Embedded Tissues
3.2 IF on Paraffin-Embedded Tissues
3.3 Whole-Mount IF
3.4 Lineage Tracing
4 Notes
References
Chapter 12: Transplantation as a Quantitative Assay to Study Mammalian Male Germline Stem Cells
1 Introduction
2 Materials
2.1 Recipient Selection
2.2 Donor Selection
2.3 Reagents
3 Methods
3.1 Donor Cell Preparation
3.2 Preparation of a Single-Cell Suspension from Neonate or Pup Testes
3.3 Preparation of Adult Testis Single-Cell Suspension
3.4 Strategies for Enrichment of SSCs
3.5 Enrichment of Spermatogonia with a Continuous Percoll Gradient
3.6 Selection of Thy1+ Cells Using MACS
3.7 Preparation of Cells from Primary Cultures of Undifferentiated Spermatogonia
3.8 Microinjection of Donor Cells into Recipient Seminiferous Tubules
3.9 Considerations for Technical and Biological Replication
3.10 Analysis of Recipient Testes for Colonies of Donor-Derived Spermatogenesis
3.11 LacZ Staining Recipient Testes
3.12 Qualitative and Quantitative Assessment of Donor-Derived Spermatogenic Colonies
3.13 Determination of SSC Purity in Fractionated Testis Cell Suspensions
4 Notes
References
Chapter 13: Mouse Fetal Germ Cell Isolation and Culture Techniques
1 Introduction
2 Materials
2.1 Isolation of PGCs by Fluorescence-Activated Cell Sorting (FACS)
2.2 Isolation of PGCs by MACS
2.3 Primary Fetal Germ Cell Culture
3 Methods
3.1 Isolation of PGCs by Fluorescence-Activated Cell Sorting (FACS)
3.2 Isolation of PGCs by MACS
3.3 Primary Fetal Germ Cell Culture
4 Notes
References
Chapter 14: Rattus norvegicus Spermatogenesis Colony-Forming Assays
1 Introduction
2 Materials
2.1 Spermatogenesis Colony-Forming Assays
3 Methods
3.1 Preparing Laminin-Coated Culture Dishes
3.2 Preparing Mouse Embryonic Fibroblast Feeder Layers
3.3 Formulating Spermatogonial Culture Media
3.4 Formulating Rat A-Single Spermatogonia Culture Media (As-SG Medium)
3.5 Formulating Spermatogonial Differentiation Medium (SD Medium)
3.6 In Vitro Spermatogenesis Colony-Forming Assay
3.7 Immunofluorescence Labeling on Spermatogonial Cultures
4 Notes
References
Chapter 15: Identification of Mouse piRNA Pathway Components Using Anti-MIWI2 Antibodies
1 Introduction
2 Materials
2.1 Recombinant MIWI2 Protein Isolation
2.2 Isolation of MIWI2-Associated piRNAs
2.3 MIWI2 Immuno-histochemistry
3 Methods
3.1 Preparation of Recombinant MIWI2 Expression Vectors
3.2 Inclusion Body Purification
3.3 Immunize Mice with Recombinant MIWI2 Inclusion Bodies
3.4 Fusion of Myeloma Cells with Immunized Mouse Lymphocytes
3.5 Immunoprecipitation of MIWI2 from Mouse Embryonic Testes
3.6 Visualization of MIWI2-Associated piRNAs
3.7 MIW2 Immuno-histochemistry in Embryonic Testis
4 Notes
References
Chapter 16: Efficient Induction and Isolation of Human Primordial Germ Cell-Like Cells from Competent Human Pluripotent Stem C...
1 Introduction
2 Materials
2.1 Reagents and Equipment
3 Methods
3.1 Maintenance of Conventional and Competent hPSCs
3.2 Conversion of Conventional and Competent hPSCs
3.3 Induction and Isolation of hPGCLCs
4 Notes
References
Index