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J Mol Biol. 2010 Sep 10;402(1):52-69. doi: 10.1016/j.jmb.2010.07.015. Epub 2010 Jul 19.

Multiplexing RMCE: versatile extensions of the Flp-recombinase-mediated cassette-exchange technology.

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  • 1Hannover Medical School OE6960 Experimental Hematology, Carl-Neuberg-Strasse 1, D-30625 Hannover,Germany.

Abstract

There are strong indications, but as yet no proof, that extended 48-bp Flp recombinase targets (FRTs) represent unique targets in all eukaryotic genomes investigated, and that recombinase-mediated cassette exchange is not hampered by the occurrence of genomic pseudo sites. This encouraged the present study in which we explore the feasibility of exchanging, in a given cell, two distinct genomically anchored cassettes, each flanked by a unique set of two heterospecific FRT sites. Mutant FRTs have to meet two major prerequisites for successful recombinase-mediated cassette exchange: (i) a self-recognition capacity comparable to a pair of FRT wild-type sites (FRTxFRT), and (ii) a negligible cross-interaction if part of a set of heterospecific sites (F'xF). We apply a two-step strategy to explore various newly created FRT spacer mutants for these properties. As a result of our screening steps, we identify combinations of sites that are successfully applied to parallel Flp-mediated genomic targeting ("multiplexing") reactions (i.e., the simultaneous exchange of two separate target cassettes in a given cell).

Copyright 2010 Elsevier Ltd. All rights reserved.

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