title: Generating transgenic frog embryos by restriction enzyme mediated integration (REMI).
abstract: Here we present a protocol for generating transgenic embryos in Xenopus laevis and Xenopus tropicalis. The method includes three steps: (1) The preparation of high-speed egg extracts, which facilitates the replacement of protamines in sperm nuclei with nucleosomes and decondenses the chromatin of sperm nuclei; (2) The isolation of sperm nuclei; and (3) The mixing of sperm nuclei, restriction enzyme, and high-speed extract in vitro, following by nuclear transplantation into unfertilized eggs to generate the transgenic embryos. This procedure generates non-mosaic transgenic embryos at high frequency and efficiency.
authors.affiliation: The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, Manchester, England, UK.
authors.forename: Shoko
authors.initials: S
authors.lastname: Ishibashi
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Kroll
authors.forename: Enrique
authors.initials: E
authors.lastname: Amaya
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D002457:Cell Extracts / Q000235:genetics; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004625:Embryo, Nonmammalian; D005260:Female; D018014:Gene Transfer Techniques; D008297:Male; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D013094:Spermatozoa / Q000166:cytology; D014461:Ultracentrifugation; D014981:Xenopus / Q000235:genetics*
chemical_list: D002457:Cell Extracts; D004262:DNA Restriction Enzymes