Using a Mammalian Virus to Create Plants for Site-Specific Transgene Insertion
Zabaronick, William John
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A novel strategy for site-specific DNA transformation of plants has been proposed and the first component of the system developed. The proposed method overcomes the limitations of current techniques by providing a specific integration site for the insertion of transgenes using features of the adeno-associated virus (AAV) life cycle. In the absence of helper virus, AAV integrates into a specific location on human chromosome 19, the AAVS1 locus. The sequence for AAV integration was introduced into the model plant Arabidopsis thaliana using Agrobacterium tumefaciens-mediated transformation. A portion of the human AAVS1 sequence, including the Rep binding site (RBS) and terminal resolution site (TRS), was cloned between T-DNA borders of the Agrobacterium Ti plasmid. The reporter gene, b-glucuronidase (GUS) was inserted proximal to AAVS1 in the plasmid for use in screening for the presence of T-DNA. In addition, it will serve as an indicator of the expression level expected for transgene inserted into AAVS1 by recombinant AAV. PCR amplification, dideoxy sequencing, GUS expression assays and genomic Southern blots were performed to examine putative transgenic plants for the presence of the AAVS1 sequence.
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