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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR TOOLS TO MINIMIZE RISK IN GENETICALLY ENGINEERED CROPS

Location: Crop Improvement & Utilization Research

Title: The phiC31 Recombinase Demonstrates Heritable Passage of Site-specific Genomic Excision in Arabidopsis

Authors
item Thomson, James
item Chan, Ronald
item Thilmony, Roger
item Ow, David

Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 15, 2009
Publication Date: February 23, 2010
Citation: Thomson, J.G., Chan, R., Thilmony, R.L., Ow, D.W. 2010. The phiC31 Recombinase Demonstrates Heritable Passage of Site-specific Genomic Excision in Arabidopsis. BioMed Central (BMC)Biotechnology. Available: doi:10.1186/1472-6750-10-17.

Interpretive Summary: Site-specific recombinases are enzymes that are capable of removing unwanted antibiotic or herbicide genes used as selectable markers during genetic engineering prior to public release. The conservative nature of the enzymes does not add or delete nucleotides thereby maintaining gene integrity. To help facilitate the freedom to operate and stimulate development of this technology our lab has developed and tested a number of novel recombinases to better control genomic engineering in plants. We present evidence demonstrating that the phiC31 recombinase can precisely remove DNA from the Arabidopsis genome and the deletion event can be inherited in progeny plants independent of the phiC31 gene.

Technical Abstract: The large serine resolvase phiC31, from the broad host range Streptomyces temperate phage, catalyzes the recombination of two differing recognition sites known as attP and attB. Minimum length sites have been determined to be 39 bp for the attP and 34 bp for the attB. In this research, the phiC31 recombinase gene was placed under control of the OXS3 promoter and introduced into Arabidopsis lines harboring a chromosomally integrated attP/attB flanked target. The phiC31 recombinase site-specifically removed the attP/attB flanked genomic DNA and the excision event was detected in subsequent generations, evenin the absence of the phiC31 gene. This establishes that germinal transmission of the excision event was obtained in the absence of the recombinase gene in the genome. The successful deletion of the genomic target sequences by this recombination system in planta, illustrates how the phiC31 recombinase can be utilized in plants to remove unneeded transgenic DNA, such as selectable markers or other introduced transgenes when they are no longer desired.

Last Modified: 8/22/2014
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