Team:BIOTEC Dresden/Project


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Overall project

Recombinases, such as Cre and Flp, have become an indispensible tool for time and tissue-specific modulation of gene expression in many model organisms. The site-specific recombinase Flp can be used to insert, delete, invert or exchange DNA fragments on plasmids as well as genomes. Flp has been critical for the generation of conditional gene knock-out models in the mouse and elsewhere, enabling detailed study of the function of a particular gene. Furthermore, recombinase-mediated cassette exchange (RMCE) allows site-specific integration of custom DNA cassettes into a prepared genome locus reliably at a high rate [1]. Much effort has been made to generate Flp variants of different activity, and to enhance its temperature stability [2, 3].

Flp recombinase catalyzes a DNA recombination event between two 34-bp Flp recognition target (FRT) sites. Occurrence of recombination events depends on random collision between both FRT sites, and not one-dimensional scanning by Flp [4]. The frequency of these collisions decreases with increasing distance between FRT sites. The quantitative relationship between this distance and FLP activity is poorly understood. This uncertainty may limit the design of experiments such as RMCE, where potentially large intervening pieces of DNA separate two FRT sites. In this project, we will characterize the activity of Flp with respect to inter-FRT site distance to complement an earlier publication [5].

[1] Qiao, J., et al., (2009). J Mol. Bio. 390, 579-594. [2] Buchholz, F., and Stewart, A.F. (2001). Nat. Biotechnol. 19, 1047-1052. [3] Santoro, S.W., Schultz, P.G. (2002). PNAS 99, 4185-4190. [4] Beatty, L.G., (1986) et al., J Mol Biol, 188, 529–544. [5] Ringrose L., et al., (1999). EMBO J. 18, 6630–6641.

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