title: Generation of Transgenic Xenopus laevis: I. High-Speed Preparation of Egg Extracts.
abstract: INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes the method for the high-speed preparation of egg extracts. Briefly, a crude, cytostatic factor (CSF)-arrested egg extract (i.e., cytoplasm arrested in meiotic metaphase) is prepared. These extracts are driven into the interphase stage of the cell cycle by addition of calcium, and high-speed centrifugation is performed to obtain a purer cytoplasmic fraction. This fraction promotes swelling of sperm nuclei, but does not promote DNA replication. By adding the egg extract to the reaction, the sperm chromatin partially decondenses, facilitating integration of plasmid DNA into the genome.
authors.affiliation: The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT,United Kingdom.
authors.forename: Shoko
authors.initials: S
authors.lastname: Ishibashi
authors.forename: Kristin L
authors.initials: KL
authors.lastname: Kroll
authors.forename: Enrique
authors.initials: E
authors.lastname: Amaya