Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates.
Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the transposon itself, the length as well as sequence of the DNA outside the transposon have significant effects on transposition. As a general rule, the closer the transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted transposons in their chromosomes. Using this assay, of the order of 10,000 independent transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.
Izsvák
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Division of Molecular Biology Centre for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Ivics
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Plasterk
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001685:Biological Factors / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D002875:Chromosomes / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D009928:Organ Specificity; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013045:Species Specificity; D013379:Substrate Specificity; D020079:Terminal Repeat Sequences / Q000235:genetics; D014162:Transfection / Q000379:methods; D014170:Transformation, Genetic / Q000235:genetics*; D019895:Transposases / Q000235:genetics / Q000378:metabolism
D001685:Biological Factors; D004251:DNA Transposable Elements; D004247:DNA; D019895:Transposases