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Development of hyperactive Sleeping Beauty transposon vectors by mutational analysis

Item Type:Article
Title:Development of hyperactive Sleeping Beauty transposon vectors by mutational analysis
Creators Name:Zayed, H. and Izsvak, Z. and Walisko, O. and Ivics, Z.
Abstract:The Sleeping Beauty (SB) transposable element is a promising vector for transgenesis in vertebrates and is being developed as a novel, nonviral system for gene therapeutic purposes. A mutagenesis approach was undertaken to improve various aspects of the transposon, including safety and overall efficiency of gene transfer in human cells. Deletional analysis of transposon sequences within first-generation SB vectors showed that the inverted repeats of the element are necessary and sufficient to mediate high-efficiency transposition. We constructed a "sandwich" transposon, in which the DNA to be mobilized is flanked by two complete SB elements arranged in an inverted orientation. The sandwich element has superior ability to transpose >10-kb transgenes, thereby extending the cloning capacity of SB-based vectors. We derived hyperactive versions of the SB transposase by single-amino-acid substitutions. These mutations act synergistically and result in an almost fourfold enhancement of activity compared to the wild-type transposase. When combined with hyperactive transposons and transiently overexpressed HMGB1, a cellular cofactor of SB transposition, hyperactive transposases elevate transposition by almost an order of magnitude compared to the first-generation transposon system. The improved vector system should prove useful for efficient gene transfer in vertebrates.
Keywords:Amino Acid Sequence, Amino Acid Substitution, Base Sequence, Catalytic Domain, Cell Line, DNA, DNA Transposable Elements, Gene Transfer Techniques, Genetic Vectors, Genetic Recombination, Molecular Sequence Data, Protein Sequence Analysis, Sequence Deletion, Transfection, Transgenes, Transposases, Animals
Source:Molecular Therapy
Publisher:Elsevier / Academic Press
Page Range:292-304
Date:1 February 2004
Official Publication:https://doi.org/10.1016/j.ymthe.2003.11.024
PubMed:View item in PubMed

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