Gene Targeting-- How The Donor Finds Its Target and How It Recombines

Gene targeting is a powerful tool for precise engineering of genomes, yet the mechanism whereby it occurs is still poorly understood. In order to understand how the T-DNA finds its target, we are applying new imaging methods to analyze several aspects of T-DNA transformation: We are determining the timing of conversion of single strand to double stranded T-DNA, the number of molecules transformed and the movement of the T-DNA itself into the nucleus. Single molecule imaging is done through the analysis of foci of GFP, YFP or RFP fused to LacI bound to LacO repeats. Another system consists of the binding of the SeqA-GFP protein to dam-methylated T-DNA. In addition we are testing the effect of facilitator proteins, e.g. AtRAD52 or AtRAD54 on the rate of gene targeting and  we are analyzing the genetic and epigenetic outcome of gene targeting events. The interpretation of these results provides some insight with regards to the gene targeting mechanism.