Germline Development Methods and Protocols

About this book

The germline is unique in mammals as it is the only cell lineage that undergoes mitosis, meiosis, and differentiation, making these cells amenable to in-depth genomic and mechanistic studies. In Germline Development: Methods and Protocols, expert researchers provide a wide-ranging collection of techniques that can be applied to the study of both the male and female germline.

As the techniques used for isolating and studying male and female germ cells are slightly dissimilar, the different technologies are grouped under two sections in this volume, namely, male germline and female germline. This collection brings the field of germline developmental studies together such that readers can find, in a single volume, techniques that allow the isolation of gonads with germ cells at different stages of development, the study of the genomics and epigenomics of germ cells, and an examination of the role of single genes in the developmental potential and function of germ 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 the avoidance of known pitfalls. Comprehensive and cutting-edge, Germline Development: Methods and Protocols serves as a comprehensive text to individuals, particularly researchers in reproductive and developmental biology, who are interested in studying cells of the germ lineage.

Contents

Part I: Male Germline
1. Isolation of Fetal Gonads from Embryos of Timed-Pregnant Mice for Morphological and Molecular Studies Yunmin Li, Teruko Taketo, and Yun-Fai Chris Lau
2. Neonatal Testicular Gonocytes Isolation and Processing for Immunocytochemical Analysis Gurpreet Manku, Monty Mazer, and Martine Culty
3. Isolation of Undifferentiated and Early Differentiating Type A Spermatogonia from Pou5f1-GFP Reporter Mice Thomas Garcia and Marie-Claude Hofmann
4. Isolation of Human Male Germline Stem Cells Using Enzymatic Digestion and Magnetic-Activated Cell Sorting Zuping He, Maria Kokkinaki, Jiji Jiang, Wenxian Zeng, Ina Dobrinski, and Martin Dym
5. Isolation and Purification of Murine Male Germ Cells Catherine Boucheron and Vanessa Baxendale
6. Preparation of Enriched Mouse Syncitia-Free Pachytene Spermatocyte Cell Suspensions Yan Wang and Martine Culty
7. Revealing Transcriptome Landscape of Mouse Spermatogonial Cells by Tiling Microarray Tin-Lap Lee, Owen M. Rennert, and Wai-Yee Chan
8. Biochemical Characterization of a Testis-Predominant Isoform of N-Alpha Acetyltransferase Alan Lap-Yin Pang
9. Identification of Novel Long Non-Coding RNA Transcripts in Male Germ Cells Tin-Lap Lee, Amy Xiao, and Owen M. Rennert
10. Methylation Profiling Using Methylated DNA Immunoprecipitation and Tiling Array Hybridization Hoi-Hung Cheung, Tin-Lap Lee, Owen M. Rennert, and Wai-Yee Chan
11. Spermatogenesis in Cryptorchidism Alexander I. Agoulnik, Zaohua Huang, and Lydia Ferguson

Part II: Female Germline
12. In Vitro Culture of Fetal Ovaries: A Model to Study Factors Regulating Early Follicular Development Shyamal K. Roy, Cheng Wang, Anindit Mukherjee, and Prabuddha Chakraborty
13. Microspread Oocyte Preparations for the Analysis of Meiotic Prophase Progression with Improved Recovery by Cytospin Centrifugation Teruko Taketo
14. In Vitro Maturation (IVM) of Porcine Oocytes Ye Yuan and Rebecca L. Krisher
15. Experimental Approaches to the Study of Human Primordial Germ Cells Andrew J. Childs and Richard A. Anderson
16. Investigating the Origins of Somatic Cell Populations in the Perinatal Mouse Ovaries Using Genetic Lineage Tracing and Immunohistochemistry Chang Liu, Melissa Paczkowski, Manal Othman, and Humphrey Hung-Chang Yao
17. DNA Methylation Analysis of Germ Cells by Using Bisulfite-Based Sequencing Methods Hisato Kobayashi and Tomohiro Kono
18. Gene Expression in Mouse Oocytes by RNA-Seq Eric Antoniou and Robert Taft

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