Chapter 2. Microinjection of exogenous nucleic acids into eggs: Ciona species
Chapter 3. Practical guide for the ascidian microinjection: Phallusia mammillata
Chapter 4. Microinjection of exogenous DNA into eggs of Halocynthia roretzi
Chapter 5. Electroporation in ascidians: history, theory and protocols
Chapter 6. The use of cis-regulatory DNAs as molecular tools
Chapter 7. Reporter Analyses Reveal Redundant Enhancers that Confer Robustness on Cis-Regulatory Mechanisms
Chapter 8. Investigating evolutionarily conserved molecular mechanisms controlling gene expression in the notochord
Chapter 9. Purification of Fluorescent Labeled Cells from Dissociated Ciona Embryos
Chapter 10. Germline transgenesis in Ciona Germline transgenesis in Ciona
Chapter 11. The enhancer trap in Ciona
Chapter 12. TALEN-based knockout system
Chapter 13. CRISPR knockouts in Ciona embryos
Chapter 14. Transgenic techniques for investigating cell biology during development
Chapter 15. Cellular Processes of Notochord Formation
Chapter 16. Morphology and Physiology of the Ascidian Nervous Systems and the Effectors
Yasunori Sasakura started his research career in Dr. Noriyuki Satoh’s laboratory in 1996. His Ph.D. thesis was about the localization of mRNAs in eggs of the ascidian Halocynthia roretzi. He then focused on developing the genetic approaches in the ascidian Ciona intestinalis. The reason is that he thought that genetics would be necessary to elucidate molecular mechanisms of maternally stored substances. He introduced transposon-mediated germ cell transgenesis and mutagenesis in this ascidian. From then on he brought many technical innovations in the study of Ciona with transgenic techniques. For example, his group has introduced transgenesis-mediated tissue/cell tracing with the photoconvertible protein Kaede, transposon-based specific knockdown of maternally transcribed RNAs, the optogenetic approach to understand neuronal functions, and genome editing techniques in Ciona with TALENs and CRISPR/Cas9.
This book comprehensively describes the transgenesis techniques and applied experimental methods in ascidians including enthusiastically developed original devices in addition to concrete examples of developmental biology studies.
Ascidians have been one of the most important model animals in developmental biology for studying molecular and cellular processes underlying formation of the chordate body plan. Transgenic techniques such as microinjection, electropolation, cis-element analysis and application, and TALENs and CRISPR/Cas9 have been developed in ascidians for more than 20 years, and now many applied methods, some of which are unique in ascidians, have been accumulated. Those extensive technological innovations, such as cell isolation, cell labeling, germ-line transformation, marker transgenic lines, and the experimental systems for studying notochord formation and nervous system, are exceptional particularly in marine invertebrates.
This book is useful for ascidian researchers to quickly access the techniques in which they are interested as well as to compare each technology to become familiar with specialized tips, and for biologists of other organisms to learn the unique techniques and ingenious attempts specific to ascidians. Providing detailed and easily understandable descriptions of techniques, the book will inspire ascidian specialists to improve their techniques, encourage anyone wanting to begin studying ascidians, and enable readers to immediately apply the techniques to the organisms they research.
