In vivo transposition of Minos, a Drosophila mobile element, in mammalian tissues.

Transposable elements have been used widely in the past 20 years for gene transfer and insertional mutagenesis in Drosophila. Transposon-based technology for gene manipulation and genomic analysis currently is being adopted for vertebrates. We tested the ability of Minos, a DNA transposon from Drosophila hydei, to transpose in mouse tissues. Two transgenic mouse lines were crossed, one expressing Minos transposase in lymphocytes under the control of the CD2 promoter/locus control region and another carrying a nonautonomous Minos transposon. Only mice containing both transgenes show excision of the transposon and transposition into new chromosomal sites in thymus and spleen cells. In addition, expression of Minos transposase in embryonic fibroblast cell lines derived from a transposon-carrying transgenic mouse resulted in excision of the transposon. These results are a first step toward a reversible insertional mutagenesis system in the mouse, opening the way to develop powerful technologies for functional genomic analysis in mammals.

Mobility assays confirm the broad host-range activity of the Minos transposable element and validate new transformation tools.

Fast and reliable methods for assessing the mobility of the transposable element Minos have been developed. These methods are based on the detection of excision and insertion of Minos transposons from and into plasmids which are co-introduced into cells. Excision is detected by polymerase chain reaction (PCR) with appropriate primers. Transposition is assayed by marker rescue in Escherichia coli, using a transposon plasmid that carries a tetracycline resistance gene and a target plasmid carrying a gene that can be selected against in E. coli. Using both assays, Minos was shown to transpose in Drosophila melanogaster cells and embryos, and in cultured cells of a mosquito, Aedes aegypti, and a lepidopteran, Spodoptera frugiperda. In all cases, mobility was dependent on the presence of exogenously supplied transposase, and both excision and transposition were precise. The results indicate that Minos can transpose in heterologous insect species with comparable efficiencies and therefore has the potential to be used as a transgenesis vector for diverse species.

Extrachromosomal transposition of the transposable element Minos occurs in embryos of the silkworm Bombyx mori.

To assess the ability of the transposable element Minos to act as a vector for genetic manipulation of the silkworm Bombyx mori, an extrachromosomal transposition assay based on three plasmids was performed. The three plasmids - helper, donor and target - were co-injected into preblastoderm embryos. Low molecular weight DNA was extracted from the embryos at the stage of blastokinesis and used to transform Escherichia coli. High frequency of transposition was observed in the presence of a helper plasmid possessing an intronless Minos transposase gene, whereas transposition did not occur in the presence of a helper plasmid with the intron-bearing transposase gene. Sequence analysis of the insertion sites showed that Minos always inserts into a TA dinucleotide. Although the insertions are distributed throughout the target gene, there was a preference for certain insertion sites. However, no consensus could be identified in the sequence flanking the target site. The results strongly suggest that the transposable element Minos has the potential to be used as a vector in the silkworm and probably in other lepidopteran insects.

Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos.

The development of efficient non-viral methodologies for genome-wide insertional mutagenesis and gene tagging in mammalian cells is highly desirable for functional genomic analysis. Here we describe transposon mediated mutagenesis (TRAMM), using naked DNA vectors based on the Drosophila hydei transposable element Minos. By simple transfections of plasmid Minos vectors in HeLa cells, we have achieved high frequency generation of cell lines, each containing one or more stable chromosomal integrations. The Minos-derived vectors insert in different locations in the mammalian genome. Genome-wide mutagenesis in HeLa cells was demonstrated by using a Minos transposon containing a lacZ-neo gene-trap fusion to generate a HeLa cell library of at least 10(5) transposon insertions in active genes. Multiple gene traps for six out of 12 active genes were detected in this library. Possible applications of Minos-based TRAMM in functional genomics are discussed.