title: Generation of Transgenic Xenopus laevis: II. Sperm Nuclei Preparation.
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 a method for the preparation of sperm nuclei from Xenopus laevis. Sperm suspensions are prepared by filtration and centrifugation, and then treated with lysolecithin to disrupt the plasma membrane of the cells. Sperm nuclei can be stored frozen in small aliquots at -80°C.
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