title: Production of transgenic Xenopus laevis by restriction enzyme mediated integration and nuclear transplantation.
abstract: Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.
authors.affiliation: The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester.
authors.forename: Enrique
authors.initials: E
authors.lastname: Amaya
authors.forename: Kristen
authors.initials: K
authors.lastname: Kroll
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics*; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D008297:Male; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D013094:Spermatozoa / Q000502:physiology; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics*
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes
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