B and T Cells Express the Ews-ERG Fusion RNA

<p>A 96-d-old mouse with both <i>Ews-ERG</i> and <i>Rag1-Cre</i> alleles was used as a source of spleen and thymus cells. Single cell suspensions of spleen cells were labelled with anti-B220 or with anti-Thy1.2 and were purified using a MoFlo preparative flow cytometer. Estimated purities were achieved of greater than 95%. cDNA was prepared from RNA extracted from sorted cells or from aliquots of unsorted populations and RT-PCR (approximately 3,400 B220+ or 6,400 Thy1.2+ cell equivalents per PCR reaction) carried out with specific <i>Pax5</i> (A), <i>CD3</i> (B) or <i>Ews-ERG</i> (C) primers. PCR reaction products were fractionated on 1% agarose gels and either stained with ethidium bromide and photographed (A and B) or gel blotted and hybridised with an <i>Ews-ERG</i> probe (C)</p

Analysis of Cell Surface Antigens of Thymomas from <i>Ews-ERG</i> Invertor Mice Using Flow Cytometry

<p>Thymus tissue was resected from mice with thymoma and FACS analysis performed to determine T cell phenotype (summarised in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-t001" target="_blank">Table 1</a>). The data in the figure show representative flow diagrams of three tumour-bearing mice (M2, M3, and M4) compared with a wild-type C57BL/6 control (wt), using anti-Thy1 (<i>y</i>-axis) plus anti-B220 (<i>x-</i>axis) antibodies or using anti-CD8 (<i>y</i>-axis) plus anti-CD4 (<i>x</i>-axis) antibodies. The three thymomas show a range of CD4 and CD8 co-expression phenotypes characterizing the thymomas generated in the <i>Ews-ERG; Rag1-Cre</i> invertor mice..</p

Incidence of Haematological Malignancy and Characteristics of Ews-ERG Invertor Mice

<p>Cohorts of 29 Ews-ERG; Rag1-Cre mice and 20 Ews-ERG control mice were monitored over a period of approximately 17 mo. Mice were culled and a post-mortem conducted when signs of ill health were observed. Leukaemia/lymphoma was established by various criteria (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#s4" target="_blank">Materials and Methods</a>). Top panel shows the survival curve of Ews-ERG; Rag1-Cre (+Cre) or Ews-ERG (−Cre) mice as a function of time (days). Bottom panel shows the histology of blood and bone marrow from leukaemic mice (M21 and M20, respectively). Blood smears were stained with May-Grünwald-Giemsa stain and photographed at 400× magnification, whilst bone marrow was photographed at 1,000×.</p

Clonality of T Cell Neoplasias in<i>Ews-ERG</i> Invertor Mice

<div><p>Genomic DNA was prepared from various tissues of invertor mice with thymomas. Genomic analysis was carried out using filter hybridisation to assess the presence of the inverted <i>ERG</i> cassette in the tumour cells (A), and to assess whether the lymphocytes involved in the tumours were clonal T (B) or B cells (C).</p> <p>(A) Inversion hybridisation autoradiograph. DNA was prepared from the thymoma and other tissues of M18, cleaved with EcoRI and hybridised to the 5′ <i>Ews</i> probe (which detects a 5-kb targeted <i>ERG</i> cassette fragment or a 6.5-kb Cre-inverted fragment). If the <i>ERG</i> cassette is inverted by Cre activity, the size of the EcoRI fragment increases from the initial targeted gene size, as indicated in the maps below the figure. The data shown are for DNA extracted from spleen (spl), thymus (thy), liver (liv), kidney (kid) or tail (ES cell DNA is used as a control). The Cre-mediated inverted band (∼6.5 kb) is evident in thymus DNA (thymoma). The summary of the data from the cohort of invertor mice is in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-t002" target="_blank">Table 2</a>. Note that despite extensive infiltration of spleen detected by histology, we cannot see the inverted band by Southern blot; this presumably reflects regional clustering of neoplastic cells in spleen. Restriction fragment sizes are represented as germ line (GL), inverted allele of <i>ERG</i> cassette (inv) and initial targeted <i>Ews</i> allele (Tgt). The organisation of the targeted <i>Ews</i> allele is indicated underneath. The hybridisation autoradiograph shows the location of <i>Ews</i> exon 7 and the initial targeted orientation (bottom) or inverted orientation of <i>ERG</i> invertor cassette after Cre-mediated recombination (top). a, acceptor splice site.</p> <p>(B) Autoradiograph showing rearrangement of T cell receptor β locus. A T cell receptor Jβ2 probe [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b40" target="_blank">40</a>] (diagrammatically shown below the autoradiograph) detects a 5-kb germ line HindIII Jβ2 band whilst V-D-Jβ2 joins in T cells result in new HindIII-sized bands depending on the nature of the rearrangement. Each of 12 thymoma DNAs that were compared showed one or two Jβ2 alleles rearranged, signifying that these tumours were clonal T cells. In some thymoma samples, there is almost complete absence of the germ line band, indicating that the thymuses of these mice are solely comprised of malignant clonal T cells. DNA sequence analysis of the V-D-J junctions of mice M2, M5, M13, and M18 showed functional V-D-J joins (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-t002" target="_blank">Table 2</a>).</p> <p>(C) Autoradiograph showing rearrangement status of immunoglobulin heavy-chain genes. A Cμ intron probe [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b41" target="_blank">41</a>] (diagrammatically shown below the autoradiograph) was used to hybridise a set of thymoma DNAs for the presence of <i>Igh</i> rearranged bands. Only two samples showed rearrangements.</p> <p>(D) Detection of Ews-Erg fusion protein in thymoma cells. Single cell suspensions were made of T cells from a normal thymus (wt) or from the thymoma of M6, protein fractionated on 4%–20% acrylamide gel, and transferred to nylon membranes. Specific proteins were detected with anti-Ews or anti-ERG antibody.</p></div

Strategy for Generating <i>Ews-ERG</i> Invertor Gene by Homologous Recombination

<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