![]() ![]() Here we report the generation of a hyperactive mutant of PBase. We have previously demonstrated that a mammalian codon-optimized version of the piggyBac transposase (PBase) mediates more efficient transposition than the original insect version, a 20-fold increase in “plasmid-to-genome” transposition ( 20), and elevated rates of chromosomal transposition ( 23). The most recent version of the Sleeping Beauty transposase (SB100) shows a marked hyperactivity compared with the original transposase ( 22). This has been successfully applied to the Sleeping Beauty transposon system. Thus, engineering the transposase is central to increasing the transposition efficiency. The transposases first bind to the IRs, then excise the DNA segment flanked by the IRs from the genome (i.e., cut) and finally reintegrate the segment into a new location (i.e., paste). The DNA transposon system consists of two components: a DNA element flanked by two terminal inverted repeats (IRs) and a transposase that catalyzes the transposon's mobilization by a “cut-and-paste” mechanism. Taking advantage of these unique characteristics, we have recently demonstrated the generation of factor-free mouse induced pluripotent stem (iPS) cells ( 21). Among them, the piggyBac transposon isolated from cabbage looper moth Trichoplusia ni is most promising because of a variety of unique characteristics, namely exhibiting the most efficient transposition in mammalian cells, the ability of the transposase to form functional protein fusions, large cargo capacity, and traceless excision, i.e., its excision restores the donor site to its pretransposon state and leaves no trace of transposon insertion ( 17 – 20). Since the generation of the Sleeping Beauty transposon, a number of transposons from different families have been reported to show active transposition in mammalian cells. Furthermore, DNA transposons hold great promise for gene therapy as nonviral vehicles ( 16). Germline transposition has accelerated the generation of mutant mice and rats ( 7 – 11), and somatic transposition has opened up numerous possibilities to conduct forward genetic screens in vivo such as cancer gene discovery in solid tumors ( 12 – 15). ![]() This pioneer work has greatly expanded the repertoire of tools for mammalian genetics. Approximately a decade ago, the first active DNA transposon in mammals, Sleeping Beauty, was reconstructed from fossilized transposon sequences found in the salmonid genome ( 6). However, their application to mammalian genetics had been hampered because of the lack of active transposons in mammals. These have been used as laboratory tools for transgenesis and insertional mutagenesis in a wide range of model organisms such as Drosophila ( 1, 2), Caenorhabditis elegans ( 3, 4), and plants ( 5). This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.ĭNA transposons are genetic elements that can mobilize from one location to an other in the host genome. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. ![]() We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. ![]() Here we report the generation of a hyperactive piggyBac transposase. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. ![]()
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