Development of hyperactive sleeping beauty transposon vectors by mutational analysis.
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.
Zayed
Hatem
H
Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse10, D-13092 Berlin, Germany.
Izsvák
Zsuzsanna
Z
Walisko
Oliver
O
Ivics
Zoltán
Z
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics* / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D011995:Recombination, Genetic / Q000235:genetics*; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection; D019076:Transgenes / Q000235:genetics; D019895:Transposases / Q000737:chemistry / Q000378:metabolism
D004251:DNA Transposable Elements; D004247:DNA; D019895:Transposases