Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. Copyright (C) 2004 John Wiley Sons, Ltd.

A study of fucoidan from the brown seaweed Chorda filum

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation.Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides.Homefucan sulfate from C.filum was shown to contain poly-a-(1-3)-fucopyranoside backbone with a high degree of branching,mainly of a-(1-2)-linked single units.Some fucopyranose residues are sulfated at O-4(mainly) and O-2 positions.Some a-(1-3)-linked fucose residues were shown by NMR to be 2-O-acetylated.The 1H and 13C NMR spectra of desulfated,deaceylated fucan were complerely assigned.THe spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit.Other fucoidan frations from C.filum have more complex carbohydrate composition and give rather complex methvlation patterns.

Knockdown and overexpression of Unc-45b result in defective myofibril organization in skeletal muscles of zebrafish embryos

We thank John Stubblefield for editing, Junling Li for the assistance in the Western blot analysis. This research was supported by a training grant from National Institutes of Health (#T32 AR07592) and a research grant MB-8713-08 from United States - Israel Binational Agriculture Research and Development Fund.