Nature Methods
2, 27 - 30 (2005)
Published online: 21 December 2004; | doi:10.1038/nmeth727
The invertor knock-in conditional chromosomal translocation mimicAlan Forster, Richard Pannell, Lesley F Drynan, Rosalind Codrington, Angelika Daser, Markus Metzler, M Natividad Lobato
& Terence H RabbittsMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Correspondence should be addressed to Terence H Rabbitts thr@mrc-lmb.cam.ac.uk Knock-in models of tumor-specific chromosomal translocations can generate lethal mutations. To circumvent this, a new conditional gene fusion model has been developed (invertor mice) and exemplified with the Ews-ERG fusion oncogene. An ERG segment, flanked by loxP sites, was knocked in to an intron of the Ews gene but in an inverted transcription orientation and lineage-specific Ews-ERG fusion created by Cre-mediated inversion. This invertor method is a completely conditional approach, applicable to any gene fusion, to emulate effects of translocations found in human cancers.
Chromosomal translocations are well-studied cancer- associated abnormalities and many breakpoints have been characterized at the molecular level1,
2. Gene fusion is a frequent outcome of translocations that lead to both leukemias and sarcomas1,
2, resulting from a break followed by rejoining during the translocation between the introns of genes. Different tumor phenotypes can result from specific versions of related fusion proteins, for instance, involving the EWS-FUS family. The EWS gene was first identified in Ewing's sarcoma by association with FLI1 and ATF1 and subsequently found in subsets of Ewing's sarcoma with either ERG or ETV1 (ref. 3) or in myxoid liposarcoma with the DDIT3 (also called CHOP) gene, analogous to FUS-DDIT3 fusion also associated with this cancer4,
5.
Conditional mouse models of chromosomal translocations are important to provide insights into the influence of gene fusions on tumorigenicity. We have previously developed two mouse models mimicking chromosomal translocations using homologous recombination in embryonic stem (ES) cells to create mutant alleles and transferred these alleles into the mouse germline. The first approach was the knock-in model6, which was exemplified by fusing the MLL gene partner MLLT3 (also called AF9) into the mouse Mll gene6. A drawback of this knock-in mouse model is that the fusion is a germline mutation, and thus cell-specific effects cannot be achieved, and in addition, the fusion protein can confer embryonic lethality7. This deficiency was obviated in the translocator model8,
9, in which de novo chromosomal translocations are created in mice10,
11, using loxP sites, to facilitate site-specific recombination by Cre recombinase10,
11,
12. In an Mll-Enl model, leukemias arose when Cre recombinase was expressed under the control of the hematopoietic stem cell gene Lmo2 (ref. 12).
The translocator approach could, in principle, be used to model any human chromosomal translocation, but if the gene orientation on the mouse chromosomes is incompatible with transcription following Cre-loxP recombination, a conditional version of the knock-in chromosomal translocation mimic strategy must be invoked. A conditional version of the original knock-in approach, using a loxP-flanked (floxed) transcription stop (loxP-STOP), has been used to avoid embryonic lethality7, but even this approach cannot always prevent low levels of read-through transcription to avoid the dominant lethal effect of fusion proteins, such as Mll-Af4 (M.M., M.N.L., A.D., A.F., R.P. and T.H.R., unpublished data). To circumvent this problem, we developed a new version of the knock-in translocation mimic, designated the invertor model. In this approach, a floxed cassette carrying a cDNA sequence with an intron segment and an acceptor splice site is knocked in to the intron of a target gene but in the orientation opposite to the direction of transcription of the target gene. RNA splicing cannot occur between the inserted cassette and the targeted gene until the cassette is inverted by expression of Cre recombinase. We have exemplified this invertor approach by modeling the EWS-ERG fusion found in some human cancers. We showed that leukemias arise in this model when the expression of Cre recombinase was controlled by the lymphocyte-specific Rag1 gene, demonstrating the efficacy of the invertor translocation mimic model as a general method for creating conditional translocation models.
Cre-loxP recombination systems are known to facilitate inversion of sequences inserted into the genome13, suggesting that knocking-in an inverted cassette flanked by loxP sites would create an absolute conditional allele in which Cre recombinase- mediated inversion was a mandatory step for protein fusion. A tumor model has been described involving inversion of an exon within the Smarcb1 (also called Snf5) gene14. The inversion approach has the additional advantage that cell-specific Cre expression will allow spatial timing for the generation of the fusion allele. We tested the invertor approach by knocking-in an ERG invertor cassette to the Ews gene (see Supplementary Methods online). The invertor cassette was constructed to comprise a short segment of intronic sequence (from intron 4 of the mouse gene Mllt2h (also called Af4), including the acceptor splice site, the ERG coding sequence usually involved in human cancer translocations, a polyadenylation site (pA) and the MC1-neo-pA gene construct for selection of homologous recombinant clones, all flanked by loxP sites with an orientation compatible for inversional recombination by Cre (Fig. 1). Detailed sequences of the components of the inversion cassette are shown in Supplementary Figs. 1 and 2 online). Gene targeting in ES cells was used to introduce the invertor cassette, cloned in an Ews genomic fragment, into the mouse Ews gene downstream of the donor splice site of exon 7 (Fig. 1a). After Cre-mediated inversion, the correct splice can occur (Fig. 1b,c); this was confirmed by transfecting targeted ES cells with a plasmid encoding Cre recombinase and amplifying the mRNA junction for sequence analysis (Fig. 1d). The targeted ES cells were injected into blastocysts, and these yielded chimeras that gave rise to heterozygous carriers of the Ews-ERG invertor allele (invertor mice).
 | | Figure 1. Strategy for generating Ews-ERG invertor gene by homologous recombination. | | |  | (a) A partial restriction map of the mouse Ews gene, including the XbaI−EcoRI fragment (indicated above the line) cloned from a 129-mouse genomic library and the invertor cassette comprising loxP-ERG-pA-MC1-neo-pA-loxP (using the human ERG cDNA sequence, Supplementary Fig. 4 online) cloned into the XbaI site just 3' of Ews exon 7. This generated the targeting vector indicated as an inset. The direction of transcription of the ERG cDNA cassette was opposite from that of the endogenous Ews gene. (b) A partial restriction map of the targeted Ews-ERG invertor allele. Homologous recombination in ES cells using the targeting vector (indicated above the line) resulted in insertion of the inverted ERG cassette just 3' of Ews exon 7 on one allele. Two probes for detecting homologous recombinants were located 5' and 3' of the Ews targeting fragment. d, donor RNA splice site (from Ews exon 7); a, acceptor RNA splice site (from the ERG invertor cassette, origin Mllt2h (Af4) intron 4; see Supplementary Fig. 2). In the targeted clone, the transcription orientation for these donor and acceptor sites is 5' 3' and 3'5', respectively. (c) Expression of Cre recombinase causes inversion of the invertor cassette to make the orientation of transcription of ERG the same as of Ews and the acceptor splice of the invertor cassette is thus upstream of ERG cDNA, allowing post-transcriptional RNA splicing from the donor site of Ews exon 7 to this acceptor site associated with ERG. R, EcoRI; K, KpnI; H, HindIII; RV, EcoRV; X, XbaI; S, SacI; B, BamHI; Bg, BglII; d, donor splice site; a, acceptor splice site. (d) Nucleotide sequence and derived protein sequence of the junction between Ews and ERG sequences in the fusion mRNA obtained by reverse transcription−polymerase chain reaction (RT-PCR) of mRNA made from Ews-ERG ES cells transiently transfected with a Cre expression vector or from spleen RNA of Ews-ERG; Rag1-cre mice (see Supplementary Methods and Supplementary Figs. 3 and 4 online). The NotI site derived from the pC2A vector is boxed. (e) Diagram showing a theoretical gene exon-intron region with an invertor cassette knock-in. As for Ews-ERG, the invertor cassette is shown in the inverse orientation for transcription (3'5') to the recipient gene. (f) After Cre recombinase expression, the invertor cassette was brought into the same orientation as the recipient gene (that is, 5'3') and thus transcription and RNA splicing can proceed to produce a fusion mRNA comprising exons of the recipient gene and of the knocked-in gene. The use of any cell-specific or inducible recombinase should generate the inversion event and allow the biological response to be studied.
 Full Figure and legend (34K) |
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We investigated the ability of the Ews-ERG fusion protein to contribute to leukemogenesis by causing the inversion of the ERG-containing cassette in lymphoid cells using Rag1-cre knock-in mice15. (Supplementary Fig. 3 online illustrates the plasmid pC2A-neo used for knocking-in the cre gene into the Rag1 gene, including the junction sequence of Rag1 and cre and other transfer cassettes suitable for knock-in homologous recombination of Cre recombinase into any gene of interest.) Ews-ERG; Rag1-cre mice developed leukemia associated with thymoma and elevated numbers of lymphocytes circulating in the blood (Fig. 2). In addition, bone marrow sections showed high levels of infiltrating lymphoblasts (Fig. 2b). Histology of spleens showed loss of normal architecture and there was invariably infiltration of leukemic lymphocytes into liver and kidney (Fig. 2b). The presence of thymomas in leukemic Ews-ERG; Rag1-cre mice suggested that the hematological malignancy in these mice involved T cells. This was confirmed by FACS analysis of T-cell surface markers. The majority of normal mouse thymic T cells express both the CD4 and CD8 surface markers, which makes them double positive cells; in addition, they express the pan-T-cell marker Thy1 (CD90) (WT, Fig. 2a). Conversely, thymomas of three invertor mice had mainly CD8+ single-positive cells (Fig. 2a).
| | |
Our work shows that the invertor model of chromosomal translocations results in an Ews-ERG fusion, which causes leukemia in mice, dependent on inversion by Cre recombinase. This chromosomal translocation mimic is applicable to any fusion gene of interest using the set of vectors described here (Fig. 1e,f). Approaches to chromosomal translocation mimics include the knock-in model6, the conditional knock-in model7, the translocator model10,
11,
12 and the invertor model described here. The new invertor mouse model is an additional approach to generate gene fusions, mimicking the consequence of chromosomal translocations, which is fully dependent of expression of Cre recombinase and inversion of a floxed intron-cDNA cassette. No fusion mRNA can be produced until inversion has taken place, as the acceptor splice site is incorrectly oriented prior to inversion. This approach is applicable to any fusion gene using the transfer cassettes described here, which are available on request (an MRC materials transfer agreement form may be downloaded at http://www2.mrc-lmb.cam.ac.uk/PNAC/Rabbitts_T/group/mta.html to expedite this). The invertor approach overcomes an inability to generate 'standard' knock-in Ews fusion alleles in ES cells and precludes any leakiness resulting from read-through transcription, which can occur with loxP-STOP conditional knock-ins because the invertor approach gives an absolute conditional allele, dependent on the Cre recombinase.
Recently, de novo translocations have been made in mice using the translocator model, which gave rise to leukemias12. This model mirrors the situation of chromosomal translocations in human cancers, as both derivative chromosomes of the translocator mice are created somatically by Cre-loxP recombination. However, translocator mice can only be made when the two genes into which loxP sites are targeted are oriented in the same direction on the relevant mouse chromosomes to gene-rate 5'3' exon fusion following translocation. The expanding sequence data available on the mouse genome has revealed that the transcription orientation of some genes, equivalent to those that fuse in human tumor-associated translocations, differs in the mouse, and this would result in dicentric chromosomes in the translocator model. In such situations, the invertor mice can be used as an alternative. Possible effects of re-inversion could be minimized in the future by using inducible, cell-specific Cre recombinase expression. Alternatively, additional recombination sites, such as frt sites for FLP recombinase activity, could be used to delete one loxP site and render the invertor cassette stable in one orientation.
Engineering mouse models of chromosomal translocations, such as knock-in, invertor and translocator mice, should be useful as a preclinical setting to test new therapeutic approaches. In particular, it is noteworthy that chromosomal translocation products are invariably intracellular; therefore, anticancer reagents must work in the intracellular milieu, and drug targeting is an important problem. Thus, one motivation for generating mouse models of chromosomal translocations is for potential use as a preclinical setting, prior to their use with patients. Several new therapeutic regimes are available, but each is at a developmental stage that requires careful experimental analysis in vivo to assess not only the efficacy but also the possible side effects. In addition, there is the all-important issue of delivering macromolecular reagents, such as siRNA or intracellular antibodies, to tumor cells. An approach to these crucial points can be made in mouse models, as a precursor to use of these reagents in humans.
Note: Supplementary information is available on the Nature Methods website.
Received 25 August 2004; Accepted 15 November 2004; Published online: 21 December 2004.
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Acknowledgments
This work was supported by the Medical Research Council. R.C. was the recipient of a Leukemia Research Fund Gordon Pillar Studentship, M.M. is the recipient of a fellowship from the German Research Foundation, and M.N.L. was the recipient of a Kay Kendall fellowship. We are very grateful to A. McKenzie for the frt-flanked PGK-neo clone. We would also like to thank G. King, A. Middleton and C. Pearce for animal husbandry and A. Lenton for the illustration work.
Competing interests statement:
The authors declare that they have no competing financial interests. |
), bone marrow section stained with hematoxylin and eosin (H&E; photographed at 1,000
