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
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
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
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
- ^ 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. 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. 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.