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Related Experiment Videos

Transgenic Xenopus laevis embryos can be generated using phiC31 integrase.

Bryan G Allen1, Daniel L Weeks

  • 1Department of Biochemistry, Bowen Science Building, University of Iowa, Iowa City, Iowa 52242, USA.

Nature Methods
|November 22, 2005
PubMed
Summary

Efficiently create transgenic Xenopus laevis embryos using bacteriophage phiC31 integrase. Insulating gene sequences with the chicken beta-globin 5' HS4 insulator ensures proper gene expression, overcoming chromatin position effects for broader applications.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • Bacteriophage phiC31 integrase facilitates DNA integration into genomes.
  • Generating transgenic organisms is crucial for biological research.
  • Xenopus laevis embryos are a valuable model system for developmental studies.

Purpose of the Study:

  • To establish a method for generating transgenic Xenopus laevis embryos using phiC31 integrase.
  • To investigate methods for achieving proper gene expression in transgenic embryos.
  • To assess the utility of insulated gene sequences for efficient transgenesis.

Main Methods:

  • Coinjection of phiC31 integrase mRNA and GFP plasmid DNA into Xenopus laevis embryos.
  • Utilizing tandem copies of the chicken beta-globin 5' HS4 insulator to flank the reporter gene.

Related Experiment Videos

  • Assessing gene expression and integration efficiency.

    • Main Results:

    • Transgenic Xenopus laevis embryos were successfully generated.
    • Integration of the reporter gene into the genome was confirmed.
    • Insulating the GFP reporter gene with the HS4 insulator was necessary to overcome silencing due to chromatin position effects and ensure appropriate promoter expression.
    • phiC31 integrase mediated efficient integration of insulated gene sequences.

    Conclusions:

    • phiC31 integrase is an effective tool for generating transgenic Xenopus laevis embryos.
    • The use of insulators is critical for achieving reliable gene expression in phiC31 integrase-mediated transgenesis.
    • This method enhances the practical application of phiC31 integrase in Xenopus and potentially other systems.