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Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system

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Abstract

The ability to perform multiple gene deletions is an important tool for conducting functional genomics. We report the development of a sequential gene deletion protocol for the filamentous fungus Aspergillus nidulans using the Cre/loxP recombinase system of bacteriophage P1. A recyclable genetic marker has been constructed by incorporating loxP direct repeats either side of the Neurospora crassa pyr-4 gene (encodes orotidine 5′-monophosphate decarboxylase) which is able to complement the A. nidulans pyrG89 mutation. This construct can be directed to delete specific genomic regions by attaching flanking sequences corresponding to the desired target. The pyr-4 marker can subsequently be eliminated by Cre-catalysed recombination between the loxP sites. The recombinase gene (cre), which has been placed under the control of the A. nidulans xlnA (xylanase A) gene promoter thus providing a means to switch on (xylose induction) or off (glucose repression) recombinase expression, has been integrated into the genome of an A. nidulans mutant strain defective in orotidine 5′-monophosphate decarboxylase activity (pyrG89). We demonstrate the effectiveness of our deletion system by sequentially deleting two genes, yellow (yA) and white (wA), involved in the synthesis of conidial pigment.

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Acknowledgments

We are grateful to Prof. C. Scazzocchio for drawing our attention to the report on the am gene in N. crassa. This work was supported by grants from the EU (QLK3-CT99-00729) and the Ministerio de Educación y Ciencia (AGL2002-01906). JVF is a recipient of a grant from the Ministerio de Educación y Ciencia.

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Authors and Affiliations

  1. Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, Apartado de Correos 73, Burjassot, 46100, Valencia, Spain

    Josep V. Forment, Daniel Ramón & Andrew P. MacCabe

  2. Departamento de Medicina Preventiva y Salud Pública, Ciencia de los Alimentos, Toxicología y Medicina Legal, Facultad de Farmacia, Universitat de València, Burjassot, 46100, Valencia, Spain

    Josep V. Forment & Daniel Ramón

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  1. Josep V. Forment
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  2. Daniel Ramón
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  3. Andrew P. MacCabe
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Correspondence to Andrew P. MacCabe.

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Communicated by C. d'Enfert

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Forment, J.V., Ramón, D. & MacCabe, A.P. Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system. Curr Genet 50, 217–224 (2006). https://doi.org/10.1007/s00294-006-0081-2

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  • DOI: https://doi.org/10.1007/s00294-006-0081-2

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