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The recognition sites for Cre recombinase called LoxP...

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Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation
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Published on: August 21, 2010

Generating transgenic frog embryos by restriction enzyme mediated integration (REMI).

Shoko Ishibashi1, Kristen L Kroll, Enrique Amaya

  • 1The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, Manchester, England, UK.

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|September 8, 2012
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Summary

We developed a new protocol to create transgenic embryos in Xenopus frogs. This efficient method uses egg extracts and sperm nuclei to generate high-quality, non-mosaic transgenic embryos.

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Area of Science:

  • Developmental Biology
  • Genetics
  • Molecular Biology

Background:

  • Generating transgenic organisms is crucial for biological research.
  • Xenopus laevis and Xenopus tropicalis are important model organisms in developmental biology.
  • Existing methods for generating transgenic Xenopus embryos can be inefficient or produce mosaic results.

Purpose of the Study:

  • To present a novel and efficient protocol for generating non-mosaic transgenic embryos in Xenopus laevis and Xenopus tropicalis.
  • To optimize the process of sperm nucleus modification and nuclear transplantation for Xenopus embryo generation.

Main Methods:

  • Preparation of high-speed egg extracts to facilitate sperm chromatin decondensation and nucleosome replacement.
  • Isolation of sperm nuclei from Xenopus sperm.
  • In vitro mixing of sperm nuclei with restriction enzymes and egg extracts, followed by nuclear transplantation into unfertilized Xenopus eggs.

Main Results:

  • The protocol successfully generates transgenic embryos in both Xenopus laevis and Xenopus tropicalis.
  • The method achieves high frequency and efficiency in producing transgenic embryos.
  • The generated transgenic embryos are non-mosaic, ensuring uniform genetic modification.

Conclusions:

  • This protocol provides a robust and efficient method for generating non-mosaic transgenic Xenopus embryos.
  • The technique is valuable for advancing research in Xenopus developmental biology and genetics.
  • The high efficiency and non-mosaic nature of the resulting embryos simplify downstream analyses.