Restriction enzyme mediated integration
Restriction enzyme mediated integration (abbreviated as REMI) is a technique for integrating DNA (linearised plasmid) into the genome sites that have been generated by the same restriction enzyme used for the DNA linearisation.[1] The plasmid integration occurs at the corresponding sites in the genome, often by regenerating the recognition sites by same the restriction enzyme used for plasmid linearisation.
Mechanism
[edit]The specific restriction enzyme cleaves the genomic DNA at random points, and generates recognition sites.[2] The DNA fragment to be inserted is linearised using the said same restriction enzyme and the mix injected into the cell followed by a successful insertion of a DNA fragment.[3]
Applications
[edit]The REMI method has been used to generate genetically modified organisms including the Saccharomyces cerevisiae, Dictyostelium discoideum and Xenopus laevis.[1][4][5]
Genome engineering
[edit]REMI has been used for large-scale transgenesis in X. laevis embryos in order to study various signaling pathways including the FGF and the Thyroid system.[3][6]
References
[edit]- ^ a b Schiestl, R. H.; Petes, T. D. (1991-09-01). "Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae". Proceedings of the National Academy of Sciences of the United States of America. 88 (17): 7585–7589. Bibcode:1991PNAS...88.7585S. doi:10.1073/pnas.88.17.7585. ISSN 0027-8424. PMC 52346. PMID 1881899.
- ^ Kessler, C.; Manta, V. (1990-08-16). "Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)". Gene. 92 (1–2): 1–248. doi:10.1016/0378-1119(90)90486-b. ISSN 0378-1119. PMID 2172084.
- ^ a b Kroll, K. L.; Amaya, E. (1996-10-01). "Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation". Development. 122 (10): 3173–3183. doi:10.1242/dev.122.10.3173. ISSN 0950-1991. PMID 8898230. S2CID 20349983.
- ^ Kuspa, A.; Loomis, W. F. (1992-09-15). "Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA". Proceedings of the National Academy of Sciences of the United States of America. 89 (18): 8803–8807. Bibcode:1992PNAS...89.8803K. doi:10.1073/pnas.89.18.8803. ISSN 0027-8424. PMC 50009. PMID 1326764.
- ^ Sparrow, Duncan B.; Latinkic, Branko; Mohun, Tim J. (2000-02-15). "A simplified method of generating transgenic Xenopus". Nucleic Acids Research. 28 (4): e12. doi:10.1093/nar/28.4.e12. ISSN 0305-1048. PMC 102591. PMID 10648800.
- ^ Turque, Nathalie; Palmier, Karima; Le Mével, Sébastien; Alliot, Caroline; Demeneix, Barbara A. (2005-11-01). "A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Xenopus Tadpoles". Environmental Health Perspectives. 113 (11): 1588–1593. doi:10.1289/ehp.7992. ISSN 0091-6765. PMC 1310923. PMID 16263516.