A Robust Marker-Free Site-Specific Gene Integration Technology

Removal of selectable marker genes and characterization of the transgene-integration locus in plants are important components of high quality transgenic lines.  Site-specific recombination (SSR) systems such as Cre-lox and FLP-FRT carry out extremely precise recombination reactions in plant genomes to either delete or integrate a defined piece of DNA, which serve as the basis of two separate applications: marker-gene removal, and site-specific gene integration.  We have combined these applications and developed an integrated technology called ‘marker-free site-specific gene integration’.  This technology allows the production of ‘marker-free’ transgenic plants containing a predictable transgene locus.  Target-specificity and impeccable precision of Cre-lox and FLP-FRT systems is expected to minimize the introduction of ‘unintended’ genetic aberrations that are often associated with the conventional plant transformation approaches or even with the targeted gene integration approaches that involve host DNA repair processes.  The developed strategy uses FLP-FRT for site-specific gene integration and Cre-lox for marker removal; although, alternative recombination systems could also be used to achieve the same result.  First, we developed an efficient site-specific gene integration method using FLP-FRT system.  The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target-FRT locus.  Transient FLP activity generated by the co-bombarded FLP gene generated the backbone-free gene circle that integrated into the target site.  Then we activated the heat-inducible cre gene to excise all marker genes from the transgene locus.  The resulting transgene locus lacked vector backbone and marker genes, and stably expressed the gene-of-interest.  The precise integration efficiency with FLP-FRT system was 20 – 30%, and the efficiency of marker removal by Cre-lox was 75 - 100%.  Thus, we developed a robust plant transformation platform for ‘marker-free site-specific gene integration’.