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United States Department of Agriculture

Agricultural Research Service

Research Project: FORAGE SYSTEMS FOR SUSTAINABLE ANIMAL PRODUCTION IN THE MID-SOUTH

Location: Forage-Animal Production Research

Title: Elimination of Marker Genes from Transformed Filamentous Fungi by Unselected Transient Transfection with a Cre-expressing Plasmid

Authors
item Florea, Simona -
item Andreeva, Kalina -
item Machado, Caroline -
item Mirabito, Peter -
item Schardl, Christopher -

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 23, 2009
Publication Date: July 2, 2009
Citation: Florea, S., Andreeva, K., Machado, C., Mirabito, P.M., Schardl, C.L. 2009. Elimination of Marker Genes from Transformed Filamentous Fungi by Unselected Transient Transfection with a Cre-expressing Plasmid. Fungal Genetics and Biology. 46:721-730.

Interpretive Summary: 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.

Technical 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.

Last Modified: 7/25/2014
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