Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid

Simona Florea; Kalina Andreeva; Caroline Machado; Peter M. Mirabito; Christopher L. Schardl

doi:10.1016/j.fgb.2009.06.010

Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid

Simona Florea, Kalina Andreeva, Caroline Machado, Peter M. Mirabito, Christopher L. Schardl

Research output: Contribution to journal › Article › peer-review

36 Scopus citations

Abstract

A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5-2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.

Original language	English
Pages (from-to)	721-730
Number of pages	10
Journal	Fungal Genetics and Biology
Volume	46
Issue number	10
DOIs	https://doi.org/10.1016/j.fgb.2009.06.010
State	Published - Oct 2009

Bibliographical note

Funding Information:
We thank Walter Hollin for technical support, and the University of Kentucky Advanced Genetic Technologies Center for DNA sequencing. Funding was provided by US Department of Agriculture Grants 2005-35319-16141, 2007-10021743, and 2008-35318-04549. Work by PMM was supported by the Gertrude Flora Ribble endowment to the University of Kentucky Department of Biology. This is Kentucky Agricultural Experiment Station publication number 09-12-074 published with the approval of the director.

Keywords

Aspergillus nidulans
Cre-lox
Epichloë
Ergot alkaloids
Fungal transformation
Gene disruption
Hygromycin B-resistance
Marker excision
Neotyphodium
dmaW
yA

ASJC Scopus subject areas

Microbiology
Genetics

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10.1016/j.fgb.2009.06.010

Cite this

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title = "Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid",

abstract = "A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum and Epichlo{\"e} festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5-2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.",

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author = "Simona Florea and Kalina Andreeva and Caroline Machado and Mirabito, {Peter M.} and Schardl, {Christopher L.}",

note = "Funding Information: We thank Walter Hollin for technical support, and the University of Kentucky Advanced Genetic Technologies Center for DNA sequencing. Funding was provided by US Department of Agriculture Grants 2005-35319-16141, 2007-10021743, and 2008-35318-04549. Work by PMM was supported by the Gertrude Flora Ribble endowment to the University of Kentucky Department of Biology. This is Kentucky Agricultural Experiment Station publication number 09-12-074 published with the approval of the director.",

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AU - Andreeva, Kalina

AU - Machado, Caroline

AU - Mirabito, Peter M.

AU - Schardl, Christopher L.

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N2 - A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5-2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.

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Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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