Validation of a novel self-excising marker by employing the beta-rec/six site-specific recombination system in the human pathogen Aspergillus fumigatus

Ref ID: 18334

Author:

Edyta Szewczyk, Michaela Dümig, Thomas Hartmann, Basem M. Jaber, Patrick Olbermann, Joachim Morschhäuser, and Sven
Krappmann

Author address:

Research Center for Infectious Diseases and Institute for Molecular Infection Biology, Julius-Maximilians-University Würzburg, Würzburg,
Germany, and Department of Biological Sciences, University of Jordan, Amman, Jordan

Full conference title:

Asperfest 8

Abstract:

Functional studies of genes often rely on methods of their manipulation, most often by means of gene targeting, such as deletion. This is achieved by
replacing the sequence of interest with a marker gene, the presence of which can be selected under specific conditions. Many cellular activities in higher
eukaryotes are often encoded bymultiple and sometimes redundant genes, making the generation of definite null mutants a difficult task. One of the crucial
problems is the limited number of selectable markers and mutations, be it nutritional or drug resistance genes. Recyclable marker modules allow repetitive
rounds of gene deletion, followed by marker rescue, making the recurring use of resistance cassettes in gene targeting tasks possible. Excision of the
selective marker also allows to avoid the potential risk of phenotypic effects caused by expression of additional heterologous genes. In aspergilli, the
Cre/lox system has recently been established, employing transient expression of the recombinase-encoding gene from an autonomously replicating plasmid,
which is later lost under non-selective culture conditions. One drawback of this method is the requirement of two rounds of transformation per gene
deletion/marker rescue event. Here, we describe functionality of a bacterial recombination system employing the small beta serine recombinase (betarec) acting on six recognition sequences in a fungal host, the human pathogen Aspergillus fumigatus. By combining a selectable marker and a tightly
controllable beta-rec expression module in the same cassette, a novel self- excising resistance marker for serial gene replacement purposes could be
validated. As further benefit, uncontrolled chromosomal rearrangements during marker excision are precluded due to the strict cis (intramolecular) action
of the beta-recombinase. This innovative cassette allows marker rescue in a more convenient manner, requiring only one transformation per
deletion/marker excision event.

Abstract Number: 12)

Conference Year: 2011

Link to conference website: NULL

New link: NULL

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