Flp / FRT system

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The Flp / FRT system is a method in genetics for removing or inserting DNA sequences . The Flp / FRT system is used, among other things, in reverse genetics and in the cassette exchange process to generate transgenic organisms .

properties

The Flp / FRT system uses the recombinase Flippase ( Flp ) to exchange DNA sequences in a site-specific manner. The recombinase Flp binds to FRT sequences and recombines them, e.g. B. in a chromosome or a plasmid . The minimal FRT sequence is (34 base pairs ):

5 ' GAAGTTCCTATTC tctagaaa G t ATAGGAACTTC 3'

The flippase binds to both ends of the FRT sequence, which are palindromic to one another on both DNA strands . In the middle of the FRT sequence there is a minimal spacer of eight base pairs (tctagaaa). On the plus strand, the flippase hydrolyses the DNA in front of the spacer, on the minus strand after the spacer.

Although different FRT sequences are known, recombination only works between two identical sequences. Some FRT sequences have an additional DNA sequence in the 5 'direction (5'-GAAGTTCCTATTCC-3') one base in front of the FRT sequence:

5 ' GAAGTTCCTATTC c GAAGTTCCTATTC tctagaaa G t ATAGGAACTTC 3'

This segment is not necessary when removing a DNA sequence (in the case of a deletion), but is necessary for an insertion, e.g. B. the RMCE cartridge exchange process. The insertion takes place with inversion of the DNA sequences. After recombination remain parts of the FRT sequences ( English FRT-Twin ). The recombination can be restricted to individual organs by tissue-specific promoters . The time of recombination can be controlled by inducible promoters (e.g. by tamoxifen or mifepristone ), e.g. B. in the expression of toxic proteins or lethal factors .

Applications

The Flp / FRT system is used to generate mutants with deletions or insertions, e.g. B. in gene knockout , in mosaic production or in the production of transgenic embryonic stem cells . It has been found in mice , Drosophila spp. who uses baker's yeast and plants .

An alternative to the Flp / FRT system is the Cre / loxP system .

Individual evidence

  1. ↑ Zhu XD, Sadowski PD: Cleavage-dependent Ligation by the FLP Recombinase . In: Journal of Biological Chemistry . 270, No. 39, 1995, pp. 23044-54. doi : 10.1074 / jbc.270.39.23044 . PMID 7559444 .
  2. ↑ Schlake T, Bode J: Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci . In: Biochemistry . 33, No. 43, 1994, pp. 12746-12751. doi : 10.1021 / bi00209a003 . PMID 7947678 .
  3. ↑ S. Turan, J. coolness, A. Schambach, C. Baum, J. Bode: Multiplexing RMCE: Versatile extensions of the Flp recombinase-mediated cassette exchange Technology . In: J. Mol. Biol. . 402, No. 1, 2010, pp. 52-69. doi : 10.1016 / j.jmb.2010.07.015 . PMID 20650281 .
  4. ^ S., Bode, J. Turan: Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications . In: FASEB J. . 25, 2011, pp. 4088-4107. doi : 10.1096 / fj.11-186940 . PMID 21891781 .
  5. ↑ S. Turan, C. Zehe, J. Kuehle, J. Qiao, J. Bode: Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. In: Genes. Volume 515, Number 1, February 2013, pp. 1-27, ISSN  1879-0038 . doi : 10.1016 / j.gene.2012.11.016 . PMID 23201421 .
  6. ↑ DA Hutcheson, G. Kardon: Genetic manipulations reveal dynamic cell and gene functions: Creating a new view of myogenesis. In: Cell cycle (Georgetown, Tex.). Volume 8, Number 22, November 2009, pp. 3675-3678, ISSN  1551-4005 . PMID 19844163 . PMC 2880528 (free full text).
  7. ↑ M. Mishina, K. Sakimura: Conditional gene targeting on the pure C57BL / 6 genetic background. In: Neuroscience research. Volume 58, Number 2, June 2007, pp. 105-112, ISSN  0168-0102 . doi : 10.1016 / j.neures.2007.01.004 . PMID 17298852 .
  8. ^ T. Lee, L. Luo: Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. In: Trends in neurosciences. Volume 24, Number 5, May 2001, pp. 251-254, ISSN  0166-2236 . PMID 11311363 .
  9. ^ R. Mortensen: Overview of gene targeting by homologous recombination. In: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]. Chapter 23 November 2006, S. Unit 23.1, ISSN  1934-3647 . doi : 10.1002 / 0471142727.mb2301s76 . PMID 19444792 .
  10. ↑ JE Belizario, P. Akamini, P. Wolf, B. Strauss, J. Xavier-Neto: New routes for transgenesis of the mouse. In: Journal of applied genetics. Volume 53, Number 3, August 2012, pp. 295-315, ISSN  2190-3883 . doi : 10.1007 / s13353-012-0096-y . PMID 22569888 .
  11. ^ NA Theodosiou, T. Xu: Use of FLP / FRT system to study Drosophila development. In: Methods (San Diego, Calif.). Volume 14, Number 4, April 1998, pp. 355-365, ISSN  1046-2023 . doi : 10.1006 / meth.1998.0591 . PMID 9608507 .
  12. ↑ HP Schweizer: Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics. In: Journal of molecular microbiology and biotechnology. Volume 5, Number 2, 2003, pp. 67-77, ISSN  1464-1801 . PMID 12736528 .
  13. ↑ H. Luo, AP Kausch: Application of FLP / FRT site-specific DNA recombination system in plants. In: Genetic engineering. Volume 24, 2002, pp. 1-16, ISSN  0196-3716 . PMID 12416298 .