<div><p>(A) The method for making invertor mice is described in detail elsewhere [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b35" target="_blank">35</a>]. In summary, an invertor cassette comprising a short intronic region with an acceptor splice site, human <i>ERG</i> cDNA sequence (shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#sg001" target="_blank">Figure S1</a>), a polyA site, and the <i>MC1neopA</i> gene all flanked by <i>loxP</i> sites (depicted by black triangles) was knocked in, using homologous recombination, into <i>Ews</i> intron 8. The transcription orientation of the targeted <i>ERG</i> cDNA invertor cassette was opposite from that of the endogenous <i>Ews</i> gene after initial homologous recombination. Germ line carrier mice of this targeted allele were crossed with <i>Cre</i>-expressing mouse strains [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b36" target="_blank">36</a>], and, as indicated in the right hand side of the diagram, the invertor cassette is turned around to create a transcription orientation identical with that of <i>Ews</i>. Thus, after transcription, a pre-mRNA is made with the donor splice site of <i>Ews</i> exon 7 adjacent to the acceptor site of the invertor cassette, allowing post-transcriptional fusion of <i>Ews</i> with <i>ERG</i> in an analogous format to that found in human sarcomas with t(21;22).</p>
<p>(B) Genomic sequence adjacent to the <i>Ews</i> exon 7 and the <i>ERG</i> invertor cassette (the derived amino acid sequence is shown in the single letter code) obtained from DNA of ES cells and of thymus after Cre-mediated inversion. The <i>Ews</i> intron 7/8 donor splice site is indicated by an arrow. The boxed sequence (
TCTAG/
CGAT) corresponds to the ligation of filled-in XbaI (<i>Ews</i> genomic) and ClaI (<i>ERG</i> invertor cassette) sites (for detailed description see [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b35" target="_blank">35</a>]). The boxed XbaI site (
TCTAGA) corresponds to the position of cloning of the mouse <i>Af4</i> intronic sequence (shaded) in the <i>ERG</i> invertor cassette. On the 3′ side of the fused XbaI-ClaI sites, there is a <i>loxP</i> site originating from the <i>ERG</i> invertor cassette (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#sg001" target="_blank">Figure S1</a>) followed by the <i>Af4</i> intron 4 (shaded) upstream of the <i>ERG</i> cDNA sequence. The <i>Af4</i> acceptor splice site is indicated by an arrow, and is followed by the <i>ERG</i> cDNA sequence. A NotI site used for cloning the amplified <i>ERG</i> sequence is boxed (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#sg001" target="_blank">Figure S1</a>) (note this sequence is non-contiguous and the dots represent a gap; the full sequence appears in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b35" target="_blank">35</a>]).
</p></div
