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

Journal of Molecular Biology
Soeren TuranJuergen Bode

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).

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Citations

Mar 29, 2013·Applied Microbiology and Biotechnology·Lydia Warth, Josef Altenbuchner
Jul 22, 2014·Nature·Mingshan XueMassimo Scanziani
Oct 15, 2014·Nature Communications·Laura ProchazkaYaakov Benenson
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Oct 29, 2013·Journal of Biotechnology·João VidigalAna P Teixeira
Jan 19, 2011·Journal of Molecular Biology·Soeren TuranJuergen Bode
Jan 18, 2014·Molecular Therapy : the Journal of the American Society of Gene Therapy·Johannes KuehleAxel Schambach
Jan 27, 2017·Proceedings of the National Academy of Sciences of the United States of America·Masaaki ToriiKazue Hashimoto-Torii
Jan 22, 2018·Biotechnology Letters·Hugo R SoaresAna S Coroadinha
Sep 6, 2011·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Soeren Turan, Juergen Bode
Oct 8, 2015·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Hong Jo LeeJae Yong Han
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Oct 21, 2021·Biology of Reproduction·Nima DehdilaniHesam Dehghani

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