A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina
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Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short (∼250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5′ and 3′ flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.
KeywordsPodospora anserina Knockout Gene replacement Homologous recombination
We greatly acknowledge Prof. Dr Christophe d’Enfert (Paris, France) for the possibility to use the E. coli strain KS272 (pKOBEG) and Prof. Dr. Axel Brakhage (Jena, Germany) and Prof. Dr. Ulrich Kück (Bochum, Germany) for helpful hints on the development of the knockout procedure. We also greatly acknowledge the P. anserina sequencing consortium coordinated at Paris, France (http://www.genoscope.cns.fr/externe/English/Projets/Projet_GA/GA.html) for making the P. anserina contigs publicly available. The experimental work is supported by a grant of the European Commission (LSHM-CT-2004-512020) to HDO.
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