Microinjection and transgenesis

The establishment of microinjection protocols about 20 years ago for cultured cells and shortly thereafter for the generation of transgenic mice by microinjection of DNA into fertilized mouse eggs greatly influ­ enced many fields of biology. Not only have the data generated using these approaches contributed to a large extent to our present under­ standing of gene regulation and cellular function of higher eukaryotic cells, but current knowledge and future developments in this area will certainly have a great impact on basic and applied research for many years to come. This laboratory manual describes the current state of the art in this research area and focuses primarily on both the experimental strategies with an extensive bibliography and the detailed procedures. A large number of studies are presently being performed and a great variety of new experimental designs are rapidly being developed. The book con­ tains protocols on injection of somatic cells as well as on injection of embryos, the use of similar equipment being a common feature. In the articles dedicated to somatic cells, full descriptions of the manual and automatic injection systems are given as well as the methods for the analysis of injected cells by video-microscopy, electron microscopy or in situ hybridizations. In addition, comprehensive protocols are given for injection experiments with very different purposes, such as to study sig­ nal transduction or microtubule dynamics.

The technique of microinjection is used to tranfer biological materials - RNA, proteins, oligonucleotides and especially cloned genes - into cultured somatic cells or embryos. Transgenic cells or animals - mice, rabbits or insects - allow the study of a wide range of cellular processes, such as gene expression, signal transduction or the cytoskeleton. More than 30 protocols on microinjection experiments and transgenesis studies developed in renowned laboratories are described in this manual. Special topics are the generation of transgenic mice using YAC, ligand-dependent site-specific recombination in embryonic stem cells, morula aggregation to generate germline chimeras as well as the analysis of injected cells.