Ismahen Ammar

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions.

The mobility of class II transposable elements (DNA transposons) can be experimentally controlled by separating the two functional components of the transposon: the terminal inverted repeat sequences that flank a gene of interest to be mobilized and the transposase protein that can be conditionally supplied to drive the transposition reaction. Thus, a DNA molecule of interest (e.g., a fluorescent marker, an shRNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be stably integrated into the genome in a regulated and highly efficient manner. Sleeping Beauty (SB) was the first transposon ever shown capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering in vertebrate species, including transgenesis, insertional mutagenesis, and therapeutic somatic gene, transfer both ex vivo and in vivo. This methodological paradigm opened up a number of avenues for genome manipulations for basic and applied research. This review highlights the state-of-the-art in SB transposon technology in diverse genetic applications with special emphasis on the transposon as well as transposase vectors currently available in the SB transposon toolbox.