1098. Duration of Expression of Sleeping Beauty Transposase by Hydrodynamic Injection of C57/BL6 Mice

Abstract

The Sleeping Beauty (SB) transposon system is extremely effective at integrating precise DNA sequences into vertebrate genomes, including those of humans. One potential use of this system is for gene therapy. The SB vector system has 2 components: 1) a transposon containing genetic cargo and 2) the catalytic transposase enzyme. In most cases the source of SB transposase has been a plasmid. This delivery strategy may allow extended periods of transposase activity that could cause deleterious effects were the transposons to be multiply remobilized. We have made several constructs to determine the duration of expression of SB transposase when delivered via a plasmid to cells in livers of mice. Three constructs contained the transposon and transposase on the same plasmid (cis constructs): pT2/mCAGGS-IDUA//Ub-SB11, pT2/mCAGGS-IDUA//MT-SB11, pT2/mCAGGS-GUSB//CMV-SB11. Another two constructs contained only a transposase gene with the transposon placed on separate plasmids (trans constructs): pT2/CAGGS-GUSB with PGK-SB11 and pT2/CAGGS-GUSB with SB11 RNA. MCAGGS is a shortened version, for driving the iduronidase (IDUA) gene, of the CAGGS promoter, which we use to drive glucuronidase B (GUSB) gene expression. We used high-pressure (hydrodynamic), tail-vein injection to deliver these constructs to the livers of C57/BL6 mice. The injections were evaluated 24hr post injection by expression assays of either the GUSB or IDUA genes. Mice will be euthanized at 6,5,4,3,2 months 6,4,2,1 weeks and 6,5,4,3,2,1 days and livers will be extracted. SB expression and functionality will be measured respectively by Western blot and excision PCR analyses. Linker-mediated PCR will be done to clone transposon insertions and integration as evidence of SB-mediated transposition events.