Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells

Abstract

Mouse has become an increasingly important organism for modeling human diseases and for determining gene function in a mammalian context. Unfortunately, transposon-tagged mutagenesis, one of the most valuable tools for functional genomics, still is not available in this organism. On the other hand, it has long been speculated that members of the Tc1/mariner-like elements may be less dependent on host factors and, hence, can be introduced into heterologous organisms. However, this prediction has not been realized in mice. We report here the chromosomal transposition of the Sleeping Beauty (SB) element in mouse embryonic stem cells, providing evidence that it can be used as an in vivo mutagen in mice.

Figure 1

A genetic-selection strategy for the detection of rare excision events. The puromycin-resistance gene driven by the PGK promoter is inactivated by the insertion of a nonautonomous SB element that separates the promoter from the coding sequence. Excision of the SB element mediated by SB transposase activates the puromycin-resistance gene. The SB element acquires novel flanking sequences when it reintegrates into the genome.

Figure 2

(a) Introduction of a single copy of the excision testing construct into the Hprt locus by gene targeting. The region of homology for the insertional-type targeting vector is indicated as gray bars. The PGK promoter and the coding sequence of the puromycin-resistance gene are shown as open boxes. The IR/DR sequences of the SB transposon are highlighted as dark arrowheads. A complete, nonautonomous SB element also is indicated in the targeting vector. The positions of the Hprt- (H) and transposon-specific (T) probes that detect excision and reinsertion events, respectively, also are indicated. All relevant BamHI sites are shown as “B” except the one that is adjacent to the excision site, which is italicized and underlined. Notice that the SB element contains a unique BamHI site approximately 1.8 kb from the end that hybridizes to the T probe. (b) Southern hybridization analysis with the H probe confirms the excision of the SB element from the testing construct. The lower intensity of the 5.5-kb fragment compared with the 7.0-kb fragment in clone 1 and clone 3 is a result of contamination with parental cells as the result of cross-feeding. The parental line has a predicted doublet consisting of a 7.0-kb and a 7.3-kb fragment. (c) Southern hybridization analysis with the T probe confirms reintegration of the excised SB element. The sizes of the bands in lanes 1, 2, 3, 4, 5, 6, 9, 11, 14, 15, 16, and 17, are variable but are all longer than 2.3 kb. No hybridization was detected in lanes 8, 10, 12, and 13. The parental fragment detected in clones 1 and 3 is due to parental cell contamination. The size marker is BstEII-digested λ-phage DNA.

Figure 3

(a) DNA sequence analysis of the donor sites after excision of the SB element from the testing construct. The four different classes of excision products are shown together with the original parental sequence. The TA dinucleotide sequence flanking the site of excision is shown in uppercase letters while the three nucleotide sequences from either ends of the SB element are shown in lowercase. The BamHI site adjacent to the site of excision and the pair of BglII sites are indicated. Both the BamHI and the BglII sites that were destroyed by the 22-bp deletion in clone #5 are boxed. (b) Schematic illustration of SB element transposition. The transposition of SB element is illustrated as a series of sequential events with events for the excision locus on the left and those for the insertion locus on the right. First, the transposase cleaves both ends of the SB element to create a 3-base 5′ overhang at the excision locus and also cleaves the TA dinucleotide at the insertion locus to create a gap with a 3′ TA overhang at both ends. After that, the excised SB element is transferred from the excision site to the new insertion site and reintegrates back into the genome. Finally, the broken ends at both the excision locus and the insertion locus are rejoined and the gaps are repaired to complete the entire transposition event.

Figure 4

Novel flanking sequences of the reintegrated SB element. The flanking sequences (14 nt from each side) of the reintegrated SB elements in clones #3, #9, and #4 are shown. The TA dinucleotides adjacent to the SB elements are shown in uppercase text while the SB elements are indicated with the last 3 nt from each end of the element in brackets. The sequence corresponding to the wild-type locus before SB insertion in clone #4 also is shown below the sequences flanking the SB element for comparison.