Evaluation of hobo element transposition in human HeLa cells
Authors:Jeonghyo Lee, Robert Rigby
Mentor:Yu Jung Kim, Professor of Biochemistry, California State University of San Bernardino
Transposons are discrete pieces of DNA that can move from one genomic location to another by a cut and paste mechanism. Since these elements generally do not depend on host factors to mediate their movement, they have been used as tools for transgenesis in many different organisms. In particular, transposons have been used in the genetic manipulation of plants and invertebrates. Only recently have transposable elements been shown to catalyze transposition in a variety of vertebrate cell lines, becoming important tools for genome engineering in vertebrates. Among the DNA transposons, the three most popular systems currently being used in vertebrate cells are: (i) Sleeping Beauty, a reconstructed Tc1/mariner element, (ii) piggyBac, a member of the piggyBac family isolated from the cabbage looper moth, and (iii) Tol2, a member of the hAT family isolated from the medaka fish. Presently, we are testing the Drosophila hAT transposon, hobo, for its ability to transpose in cultured human HeLa cells. Our objective is to determine if hobo could be used as a tool for genetic manipulations in vertebrates. For this study, we constructed two plasmids: a donor plasmid containing the hobo transposon bearing a kanamycin/neomycin resistance cassette and a helper plasmid containing the transposase gene placed under control of the cytomegalovirus promoter. We next measured the efficiency of hobo transposition from the donor plasmids to the HeLa genome by counting the number of G418-resistant cells following transfection with variable concentrations of donor plasmid (10-500 ng) and helper plasmid (0-500 ng). Based on preliminary results, we found the number of G418-resistant colonies increased with respect to increasing amounts of hobo donor and helper plasmids. To confirm whether this increase was due to transposition, we are currently performing plasmid rescue experiments to recover transposon-chromosomal junction sequences using genomic DNA isolated from G418-resistant HeLa cell clones.