Click here to close Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly. We suggest using a current version of Chrome, FireFox, or Safari.
XB-ART-44389
CSH Protoc September 1, 2007; 2007 pdb.prot4840.

Generation of Transgenic Xenopus laevis: III. Sperm Nuclear Transplantation.



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. 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 nuclear transplantation in Xenopus laevis. Permeabilized sperm nuclei are incubated briefly with linearized plasmid DNA, after which egg extract and a small amount of restriction enzyme are added. The egg extract partially decondenses the chromosomes, and the restriction enzyme stimulates recombination by creating double-strand breaks, facilitating integration of DNA into the genome. Diluted nuclei are transplanted into unfertilized eggs. Because the transgene integrates into the genome prior to fertilization, the resulting transgenic embryos are not chimeric and there is no need to breed to the next generation in order to obtain nonmosaic transgenic animals.

PubMed ID: 21357173
Article link: CSH Protoc
Grant support: [+]


Xenbase: The Xenopus Model Organism Knowledgebase.
Version: 4.15.0
Major funding for Xenbase is provided by grant P41 HD064556