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In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression
Published on: March 29, 2019
Group II intron-based gene targeting reactions in eukaryotes
Marta Mastroianni1, Kazuo Watanabe, Travis B White
1Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of America.
Plos One
|September 5, 2008
Summary
Mobile group II introns can now be used for gene targeting in eukaryotes. Researchers optimized retrohoming efficiency in eukaryotic nuclei by increasing Mg(2+) concentrations, enabling applications in higher organisms.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Mobile group II introns facilitate site-specific DNA insertion via retrohoming.
- This process involves intron-encoded proteins and RNA within a ribonucleoprotein particle (RNP).
- Group II introns have been engineered as bacterial gene targeting vectors but not yet efficiently in eukaryotes.
Purpose of the Study:
- To investigate the efficiency of group II intron RNPs in eukaryotic gene targeting.
- To optimize retrohoming conditions for enhanced integration and DNA repair in eukaryotic systems.
- To assess the potential of group II introns for gene targeting in various eukaryotic model organisms.
Main Methods:
- Utilized a plasmid-based Xenopus laevis oocyte microinjection assay.
- Manipulated Mg(2+) concentrations to optimize RNP integration efficiency.
- Analyzed integration products for double-stranded DNA characteristics and assessed homologous recombination stimulation.
- Tested targeting reactions in zebrafish and Drosophila embryos, including chromosomal targets.
Main Results:
- Group II intron RNPs efficiently integrate into eukaryotic target DNA with increased Mg(2+), reaching up to 38% of plasmid targets.
- Integration products confirm second-strand DNA synthesis by host enzymes.
- RNPs stimulate homologous recombination up to 4.8% of target sites.
- Chromatinization inhibits targeting, but DNA replication in zebrafish and Drosophila embryos mitigates this effect, allowing efficient integration into plasmid and chromosomal sites.
Conclusions:
- Demonstrated efficient, Mg(2+)-dependent group II intron integration and gene targeting in eukaryotic nuclei.
- Showcased successful application in Xenopus, zebrafish, and Drosophila, paving the way for higher organisms.
- Established a foundation for developing group II intron-based gene targeting tools for eukaryotes.
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