Lacal, Juan Carlos, Perona, Rosario, Feramisco, James (Eds.)
Softcover reprint of the original 1st ed. 1999, 248 p.
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Microinjection of macromolecules into living cells has proved to be a powerful technique for the functional analysis of gene products in living somatic cells. This approach is useful for the introduction of functional proteins, genes, inhi bitors of enzyme activities and antibodies into living cells (Ansorge, 1982). Types of cells into which these molecules can be introduced include primary and established cell lines, fIbroblasts and cells of various tissue types, including contractile cardiac cells. Some of the advantages of microinjection over other methods of introducing biological materials into cells (Celis, 1984; Graessmann et aI. , 1980; Pepperkok et aI. , 1988) are as follows: (1) Economy of the material; 13 15 microinjection delivers small volumes (10- _10- 1) directly into specifIc cells which means that the amount of material needed is much less than would be required for other approaches (e. g. , scrape loading). (2) Damage to the cell ap pears to be much less than with other methods as evidence by stress re sponses, etc. (3) There is much less limitation on the nature of the material in jected; for example, nucleic acids, proteins, large and small molecules can all be introduced with little alteration of the technique. (4) Temporal experiments can be performed where the response at a given time to a particular reagent may be monitored.
1. Needle Microinjection: A Brief History.- 2. Microinjection of Macromolecules into Mammalian Cells in Culture.- 3. The Use of Plasmid Microinjection to Study Specific Cell Cycle Phase Transitions.- 4. Combining Microinjection with Microanalytical Methods to Investigate Regulation of Metabolic Pathways.- 5. Import of Stably Folded Proteins into Peroxisomes.- 6. Study of the Function of Microtubule Proteins.- 7. Cytoskeleton Regulation by Rho Proteins.- 8. Cytoskeletal Regulation by Rho-p21 Associated Kinases: Analysis of ROK and PAK Function by Plasmid Microinjection.- 9. Use of Microinjection to Study Apoptosis and its Prevention.- 10. Role of the InsP3 Receptor in Intracellular Ca2+ Release and Ca2+ Entry.- 11. Microinjection of Xenopus laevis Oocytes: A Model System.- 12. Xenopus laevis Oocytes as a Model for Studying the Activation of Intracellular Kinases.- 13. Signal Transduction in Xenopus laevis Oocytes by Ras Oncoproteins and Lipid Metabolites.- 14. The Multiple Roles of Mos During Meiosis.- 15. Use of the Xenopus Oocyte System to Study RNA Transport.- 16. Functional Expression of G Protein-Coupled Receptors in Xenopus laevis Oocytes.- 17. Expression of Glucose Transporters in Xenopus laevis Oocytes: Applications for the Study of Membrane Transporter Function and Regulation.- 18. DNA Injection into Xenopus laevis Embryos as a Tool to Study Spatial Gene Activity.- 19. Transformation of Nematodes by Microinjection.- Guide to Solutions.- Guide to Protocols.- Troubleshooting Guide.