Germ Cell Protocols

The study of germ cells has undergone enormous advances in recent years and has entered into an explosive phase of new discoveries with the introd- tion of transgenic technologies and nuclear cloning. Basic knowledge and te- niques developed for lower vertebrate and invertebrate systems have facilitated the study of higher vertebrates, including humans. Many experiments that have first been performed on lower vertebrates provided the tools and strategies that could later be applied to other less readily available mammalian systems. The discovery of centrosomes in ascidians and sea urchin eggs now benefits st- ies of fertility and infertility in mammals including humans. External in vitro fertilization, now a common technique in assisted fertilization has only been possible as a result of numerous studies in lower systems in which external fertilization is natural. Egg activation, first explored in sea urchin and asc- ian eggs, now benefits cloning efficiency in farm and domestic animals. Gene manipulations and molecular methods have added to the possibilities of p- ducing live offspring with enormous biomedical, ecological, and economic implications. All sexually reproducing organisms produce primordial germ cells, a small population of cells that differentiate into gametes of either sex that carry to- potency, an ability to develop into an entire new organism. The two volumes on germ cells combine techniques in a variety of different systems and have selected those systems that have provided landmarks in advancing our kno- edge on germ cells.

Includes supplementary material: sn.pub/extras

Klappentext
Germ cell research has produced enormous advances in recent years and more recently has entered into an explosive phase of new discoveries with the introduction of transgenic technologies and nuclear cloning. In Germ Cell Protocols, expert laboratory investigators describe in step-by-step detail a series of powerful techniques for the molecular and genetic analysis of germ cells in a variety of different reproductive systems. Chosen for their proven success in advancing our understanding of germ cells, these methods cover sperm and egg activation, motility, fertilization, nuclear development, nuclear cloning, the molecular characterization of specific events, the imaging of cell structures, and the cryopreservation of germ cells. Volume 2: Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning focuses on molecular egg analysis, live egg imaging, nuclear cloning, oocyte cryopreservation, and nuclear transfer. Highlights include the identification and characterization of Oct-4, germline chromatin silencing by RNAi, cDNA subtraction and cloning in the field of trophoblast/placental development, and selective ablation. There are also methods for PCR-based cloning, differentiating embryonic stem cells into embryonic bodies, live imaging of mammalian embryos using multiphoton microscopy, and the cloning of pig, cattle, rabbit, and mouse oocytes. Each readily reproducible protocol is described in step-by-step detail and contains an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. An accompanying earlier volume, Sperm and Oocyte Analysis, focuses on sperm cells, oocyte analysis, oocyte maturation, fertilization, and preparation techniques.
Comprehensive and cutting-edge, Germ Cell Protocols offers both novice and established researchers a gold-standard collection of hard-to-find methods for high impact research, diverse procedures that are easy-to-follow,well-illustrated, and allow a cross-species transfer of knowledge from lower vertebrates to higher mammalian systems.

Inhalt
Activity of the Germline-Specific Oct4-GFP Transgene in Normal and Clone Mouse Embryos.- Analysis of Germline Chromatin Silencing by Double-Stranded RNA-Mediated Interference (RNAi) in Caenorhabditis elegans.- cDNA Subtraction and Cloning in the Field of Trophoblast/Placental Development.- PCR-Based Cloning and Differential Screening of RNAs from Xenopus Primordial Germ Cells.- Differentiating Embryonic Stem Cells into Embryoid Bodies.- The Derivation of Murine Embryonic Stem and Embryonic Germ Cells by Selective Ablation.- Using Multiphoton Excitation to Explore the Murky Depths of Developing Embryos.- Dual-Channel Confocal Ratioing of Calcium Dynamics in Living Eggs and Oocytes.- A Method for Producing Cloned Pigs by Using Somatic Cells as Donors.- Pronuclear Microinjection of Mouse Zygotes.- Parthenogenesis and Nuclear Transfer in Rabbit Oocytes.- Nuclear Transfer in Cattle.- In Vitro Fertilization and Embryo Transfer in Felids.- Production of Chimeric Chickens.- Production of Haploid and Diploid Androgenetic Zebrafish.- Construction and Detection of Fluorescent, Germline Transgenic Zebrafish.- Production of Zebrafish Germline Chimeras from Cultured Cells.- Studying Nuclear Reprogramming with Cell Hybrids.- Cytoplasmic Transfer in Mammalian Eggs and Embryos.- Conventional Technologies for Cryopreservation of Human Oocytes and Embryos.- Vitrifying and Warming of Human Oocytes, Embryos, and Blastocysts.- Microfluidic Removal of Cumulus Cells from Mammalian Zygotes.- Mammalian Embryo Culture in a Microfluidic Device.- Application of Alginate Gels to the Study of Mammalian Development.- Using TUNEL in Combination with an Active Caspase-3 Immunoassay to Identify Cells Undergoing Apoptosis in Preimplantation Mammalian Embryos.