The most potent technique to study cell function is the specific tranfer of genetic material into a single cell. Several functions can than be analyzed in the resulting transgenic cell or organism. The method of microinjection and transgenesis is currently used in many cell biological laboratories.

Klappentext

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.

Inhalt

Part 1 Strategies and Protocols for Microinjection Studies Using Somatic Cells in Culture.-1 Protocols for the Manual and Automatic Microinjection of Somatic Cells in Culture and for the Analysis of Microinjected Cells, Angel Cid-Arregui, Carla Santana, Miriam Guido, Nestor Morales-Peza, Maria Victoria Juarez, Roger Wepf, and Alejandro Garcia-Carranca.-2 Automated Computer-Assisted Microinjection into cultured somatic cells, Wilhelm Ansorge and Rainer Saffrich .-3 Immunofluorescence and Immunoelectron Microscopy in Microinjected and Transfected Cultured Cells, Gustafo Egea, Teresa Babia, Roser Pagan, Roser Busca, Inmaculada Ayala Ferran Vallerrama, Manuel Reina, and Senen Vilaro.-4 Videomicroscopy of Living Microinjected Hippocampal Neurons in Culture, Frank Bradke and Carlos G. Dotti .-5 Preparation, Purification, and Analysis of Synthetic Oligonucleotides Suitable for Microinjection Experiments, Ramon Güimil Garcia and Ramon Eritja .-6 Colorimetric in Situ Hybridization, Jaime Berumen .-7 Manual Microinjection: Measuring the Inhibitory Effect of HIV-1-Directed Hammerhead Ribozymes in Tissue Culture Cells, Robert Hormes and Georg Sczakiel .-8 Cellular Microbiochemistry, Rainer Pepperkok, Olaf Rosorius, and Jochen Scheel.-9 A New Strategy for Studying Microtubule Transport and Assembly During Axon Growth, Peter W. Baas . Part 2 Strategies and Protocols for Microinjection Experiments Using Embryos.-10 The Use of Microinjection to Study Signal Transduction in Mammalian Cells, Serge Roche and Sara A. Courtneidge. -11 Supply and Husbandry of Mice for Transgenic Science, Jane Morrell .-12 Mouse Male Germ Cells in Culture: Toward a New Approach in Transgenesis?, Minoo Rassoulzadegan, Julien Sage, and Valrie Grandjean.-13 Sperm-Mediated Gene Transfer, Marialuisa Lavitrano, Valentina Lulli, Barbara Maione, Sabina Sperandio, and Corrado Spadafora .-14 Application of Ligand-Dependent Site-Specific Recombination in ES Cells, Pierre-Olivier Angrand, Catherine P. Woodroofe, andA. Francis Stewart.-15 A Modified Protocol for the Generation of Transgenic Mice and Freezing of Mouse Embryos, Carol Murphy, Luis Martin-Parras, and Ulrich Rüther.-16 The Use of Morula Aggregation to Generate Germline Chimeras from Genetically Modified Embryonic Stem Cells, Miguel Torres .-17 The Generation of Transgenic Mice with Yeast Artificial Chromosomes, Holger Hiemisch, Thorsten Umland, Llus Montoliu, and Günther Schütz.-18 Following Up Microinjected mRNAs: Analysis of mRNA Poly(A) Tail Length During Mouse Oocyte Maturation, Fatima Gebauer .-19 Mouse Preimplantation Embryos and Oocytes as an In Vivo System to Study Transcriptional Enhancers, Sadhan Majumder .-20 The Use of Mouse Preimplantation Embryos for the Identification of DNA-Binding Proteins Essential in Early Development, Valerie Botquin, Jörg R. Schlehofer, Angel Cid-Arregui .-21 Application of Transgenes and Transgenic Mice to Study Gene Activity from the Oocyte to the Early Embryo, Sylvie Forlani, Lucile Monfort, and Jean-Franois Nicolas .-22 Transitory Transgenic Analysis as an In Vivo System to Study Promoter Regulatory Elements, Diana Escalante-Alcalde and Luis Covarrubias.-23 Transgenic Analysis of Embryonic Gene Expression Using LacZ as a Reporter, Catherine E. Ovitt, Young Il Yeom, and Hans R. Schöler.-24 Autonomous Cell Labeling Using a laacZ Reporter Transgene to Produce Genetic Mosaics During Development, Luc Mathis and Jean-Franois Nicolas .-25 Multiple Developmental Functions of the Zinc Finger Gene Krox-20 Are Revealed by Targeted Insertion of the E. coli lacZ Sequence, Sylvie Schneider-Maunoury, Piotr Topilko, Tanja Seitanidou, Giovanni Levi, Paula Murphy, and Patrick Charnay.-26 Transgenic Mouse Strategies in Virus Research, Angel Cid-Arregui, Nestor Morales-Peza, Prasert Auewarakul, Lutz Gissmann, Maria Jose Garcia-Iglesias, Maria Victoria Juarez, and Alejandro Garcia-Carranca.-27 Isolation of Mutated Genes from Transgene Insertion Sites, Miriam H. Meisler, James Galt, John S.