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. 2001 Sep 25;98(20):11474-8.
doi: 10.1073/pnas.201392398. Epub 2001 Sep 18.

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

L Zagoraiou  1 , D Drabek, S Alexaki, J A Guy, A G Klinakis, A Langeveld, G Skavdis, C Mamalaki, F Grosveld, C Savakis
Affiliations

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

L Zagoraiou et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Minos-derived vectors. Minos inverted terminal repeats are shown as thick, solid arrows. Open blocks outside these arrows indicate the sequences flanking the original Minos element in the D. hydei genome. Arrowheads indicate the positions of primers used to detect Minos excisions. Small arrows indicate the direction of transcription of the GFP and transposase genes. Solid bars represent fragments used as probes. All other features are described in Materials and Methods.
Figure 2
Figure 2
Tissue-specific expression of Minos transposase in transgenic mice. Northern blot analysis of thymus, spleen, and kidney RNA isolated from TM2/+ mice (40-h exposure). Control RNA is from thymus of a nontransgenic mouse. The lower image shows the signal obtained on rehybridization of the same filter with a mouse actin probe (3-h exposure).
Figure 3
Figure 3
Transposase-dependent, tissue-specific excision of a Minos transposon in mice. Oligonucleotide primers flanking the transposon were used for PCR, and the products were analyzed by agarose gel electrophoresis. (Left) Transposase-dependent excision in the thymus. Lanes: 1, nontransgenic DNA; 2, TM2/+; 3–7, MCG/+; 8–12, MCG/+ TM2/+. (Right) Excision in various tissues of transposase-expressing mice. Lanes: 1, 3, 5, 7, 9, and 11, from MCG/+ mice; 2, 4, 6, 8, 10, and 12, from MCG/+ TM2/+ mice; 1 and 2, thymus; 3 and 4, spleen; 5 and 6, liver; 7 and 8, kidney; 9 and 10, brain; 11 and 12, muscle; 13, no DNA added. Amplification of the entire transposon is not detectable under the PCR conditions used. The lower band present in most lanes probably represents primer or primer extensions during the PCR.
Figure 4
Figure 4
Footprints left behind at chromosomal sites after Minos excision. DNA was extracted from thymus and spleen of a double-transgenic mouse (Upper) or from an embryonic fibroblast cell line from a MCG/+ mouse after transfection with a transposase-expressing plasmid (Lower) and used as template for PCR with the flanking primers. PCR-amplified bands were cloned, and 32 clones (19 from thymus and spleen and 13 from fibroblast cells) were sequenced. TA is the target-site duplication. Nucleotides in red correspond to the ends of the transposon terminal repeats; nucleotides in blue are of unknown origin. The flanking nucleotides and TA repeats are aligned.
Figure 5
Figure 5
FISH analysis of Minos transpositions in thymus and spleen. Chromosomes were stained with 4′,6-diamidino-2-phenylindole. A1 and A2 are from the same MCG/+ metaphase nucleus, probed with a GFP and a telomere 14 specific probe, respectively. B–I are nuclei from thymus or spleen of MCG/+ TM2/+ mice. Nuclei in B1, C1, and D–I are probed with the GFP probe. B2 and C2 are the same nuclei as B1 and C1, respectively, probed with the telomere 14 specific probe. Yellow arrowheads indicate the original integration site of the transposon transgene, near the telomere of chromosome 14. Green arrowheads indicate the telomeres of chromosome 14. Red arrowheads indicate transposition events.
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