The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that cuts the transposon from a plasmid and pastes it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts.
Bibliographical noteFunding Information:
We thank Scott McIvor, Lalitha Belur, and Xianzheng Zhou from the Center for Genome Engineering of the University of Minnesota, and Laurence Cooper from M.D. Anderson Cancer Center of University of Texas for helpful discussions and Brenda Koniar from Resource Animal Resources and Nathan Hoekstra, Ann Clifford, and Anita Lowy from the Center for Genome Engineering of the University of Minnesota for assistance with experiments. This project was supported by P01HD32652 and R01DK082516 from the National Institutes of Health to P.B.H. and funding from Center for Genome Engineering at the University of Minnesota. P.B.H. has equity in Discovery Genomics, Inc., a small biotech company that receives funding from the NIH to explore the use of the Sleeping Beauty Transposon System for gene therapy.
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