Retroviral Insertion Mutagenesis in Mice as a Comparative Oncogenomics Tool to Identify Disease Genes in Human Leukemia
Authors: Ivo P Touw, Stefan J Erkeland
Journal: Molecular Therapy (2006)

Retroviral insertion mutagenesis has recently received much attention because of its adverse effects in the application of retroviral vector-based gene therapy, resulting in leukemia in certain patients. At the same time, retroviral mutagenesis in mice is being considered a powerful forward genetic strategy to identify disease genes involved in cancer. The publication of the mouse genome sequence and the development of high-throughput genomic approaches have given a further boost to this rapidly evolving field. The increasing numbers of new potential oncogenes identified in retroviral screens have given a valuable basis for a better understanding of cancer related pathways in mice. Important challenges that now lie ahead of us are (i) to determine the relevance and causal relationship of these genes with various types of human cancer (ii) to develop strategies to identify tumor suppressor genes on a large scale, (iii) to place the disease genes into regulatory networks to better understand their role in the complex pathogenesis of cancer, and (iv) to determine their value for diagnosis refinement and therapeutic target intervention in human disease. In this review, we will give a brief update of the current state-of-the-art and thoughts concerning these issues. We will specifically focus on the value of employing retroviral insertion mutagenesis in mice and gene expression profiling in man in the context of acute myeloid leukemia. Retroviral insertion mutagenesis has recently received much attention because of its adverse effects in the application of retroviral vector-based gene therapy, resulting in leukemia in certain patients. At the same time, retroviral mutagenesis in mice is being considered a powerful forward genetic strategy to identify disease genes involved in cancer. The publication of the mouse genome sequence and the development of high-throughput genomic approaches have given a further boost to this rapidly evolving field. The increasing numbers of new potential oncogenes identified in retroviral screens have given a valuable basis for a better understanding of cancer related pathways in mice. Important challenges that now lie ahead of us are (i) to determine the relevance and causal relationship of these genes with various types of human cancer (ii) to develop strategies to identify tumor suppressor genes on a large scale, (iii) to place the disease genes into regulatory networks to better understand their role in the complex pathogenesis of cancer, and (iv) to determine their value for diagnosis refinement and therapeutic target intervention in human disease. In this review, we will give a brief update of the current state-of-the-art and thoughts concerning these issues. We will specifically focus on the value of employing retroviral insertion mutagenesis in mice and gene expression profiling in man in the context of acute myeloid leukemia. Oncogenic retroviruses are divided in two major categories, the acute and slow-transforming retroviruses. The acute transforming retroviruses induce polyclonal tumors with a short latency of usually approximately 2–3 weeks after infection of the host and are able to transform cells in vitro.1Jonkers J Berns A Retroviral insertional mutagenesis as a strategy to identify cancer genes.Biochim Biophys Acta. 1996; 1287: 29-57PubMed Google Scholar The oncogenic activity of these viruses is dependent on the transforming sequences present in the viral genome. These defective viruses have exchanged viral sequences by recombination with cellular proto-oncogenes. Examples of acute transforming tumor viruses are avian myeloblastosis virus containing the viral (v)-myb gene, derived from cellular (c)-myb,2Lipsick JS Wang DM Transformation by v-Myb.Oncogene. 1999; 18: 3047-3055Crossref PubMed Scopus (100) Google Scholar avian MC29 virus containing v-myc,3Alitalo K et al.Nucleotide sequence to the v-myc oncogene of avian retrovirus MC29.Proc Natl Acad Sci USA. 1983; 80: 100-104Crossref PubMed Scopus (102) Google Scholar, 4Lee CM Reddy EP The v-myc oncogene.Oncogene. 1999; 18: 2997-3003Crossref PubMed Scopus (39) Google Scholar and Abelson murine leukemia virus (MLV) expressing v-abl.5Shore SK Tantravahi RV Reddy EP Transforming pathways activated by the v-Abl tyrosine kinase.Oncogene. 2002; 21: 8568-8576Crossref PubMed Scopus (32) Google Scholar Slow-transforming retroviruses induce mono- or oligoclonal tumors in mice with latencies of at least 3 months and do not efficiently transform cells in vitro.1Jonkers J Berns A Retroviral insertional mutagenesis as a strategy to identify cancer genes.Biochim Biophys Acta. 1996; 1287: 29-57PubMed Google Scholar, 6Wolff L Contribution of oncogenes and tumor suppressor genes to myeloid leukemia.Biochim Biophys Acta. 1997; 1332: F67-F104PubMed Google Scholar These viruses do not contain proto-oncogenes but cause transformation as a result of proviral integration into the host genome. Leukemias induced by slow-transforming retroviruses are characterized by a preleukemic phase in which millions of infection events may occur. Some cells with virus insertions may gain an increased growth potential, but multiple additional events are required for full transformation. Because these retroviruses are replication competent, precursor cells will be infected multiple times, which may result in deregulation of additional genes involved in tumor progression. Only cells that contain the right combination of oncogenic events will have a malignant potential and will grow out to form a tumor. The development of leukemias caused by different MLVs is to a large extent dependent upon the viral transcriptional control region (U3 region) located within the long terminal repeat (LTR).7Speck NA et al.Mutation of the core or adjacent LVb elements of the Moloney murine leukemia virus enhancer alters disease specificity.Genes Dev. 1990; 4: 233-242Crossref PubMed Scopus (131) Google Scholar In this region, multiple cell-type-specific regulatory factors can bind that influence transcriptional activity and virus replication.8Barat C Rassart E Members of the GATA family of transcription factors bind to the U3 region of Cas-Br-E and graffi retroviruses and transactivate their expression.J Virol. 1998; 72: 5579-5588PubMed Google Scholar, 9Barat C Rassart E Nuclear factors that bind to the U3 region of two murine myeloid leukemia-inducing retroviruses, Cas-Br-E and Graffi.Virology. 1998; 252: 82-95Crossref PubMed Scopus (6) Google Scholar, 10Paquette Y Kay DG Rassart E Robitaille Y Jolicoeur P Substitution of the U3 long terminal repeat region of the neurotropic Cas-Br-E retrovirus affects its disease-inducing potential.J Virol. 1990; 64: 3742-3752PubMed Google Scholar, 11Yoshimura FK Wang T Role of the LTR region between the enhancer and promoter in mink cell focus-forming murine leukemia virus pathogenesis.Virology. 2001; 283: 121-131Crossref PubMed Scopus (3) Google Scholar, 12Yoshimura FK Wang T Cankovic M Sequences between the enhancer and promoter in the long terminal repeat affect murine leukemia virus pathogenicity and replication in the thymus.J Virol. 1999; 73: 4890-4898PubMed Google Scholar, 13Yoshimura FK Davison B Chaffin K Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner.Mol Cell Biol. 1985; 5: 2832-2835Crossref PubMed Scopus (25) Google Scholar, 14Nishigaki K et al.Analysis of the disease potential of a recombinant retrovirus containing Friend murine leukemia virus sequences and a unique long terminal repeat from feline leukemia virus.J Virol. 2002; 76: 1527-1532Crossref PubMed Scopus (20) Google Scholar Thus, the T-cell lymphoma-inducing virus Moloney murine leukemia virus, SL3-3, and MCF13 show the highest transcriptional activity in T cells, whereas the erythroleukemia-inducing Friend virus is more active in erythroid cells.11Yoshimura FK Wang T Role of the LTR region between the enhancer and promoter in mink cell focus-forming murine leukemia virus pathogenesis.Virology. 2001; 283: 121-131Crossref PubMed Scopus (3) Google Scholar, 15Brightman BK Li QX Trepp DJ Fan H Differential disease restriction of Moloney and Friend murine leukemia viruses by the mouse Rmcf gene is governed by the viral long terminal repeat.J Exp Med. 1991; 174: 389-396Crossref PubMed Scopus (4) Google Scholar, 16Pantginis J Beaty RM Levy LS Lenz J The feline leukemia virus long terminal repeat contains a potent genetic determinant of T-cell lymphomagenicity.J Virol. 1997; 71: 9786-9791PubMed Google Scholar, 17Uittenbogaart CH et al.Thymic dendritic cells are primary targets for the oncogenic virus SL3-3.J Virol. 1998; 72: 10118-10125PubMed Google Scholar, 18Kim R et al.Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas.J Virol. 2003; 77: 2056-2062Crossref PubMed Scopus (93) Google Scholar This cell-type specificity can be employed to identify genes that are specifically involved in the pathogenesis of a distinct cytological subtype of leukemia. The Graffi-1.4 virus induces mainly acute myeloid leukemia (AML).19Rassart E et al.Molecular analysis and characterization of two myeloid leukemia inducing murine retroviruses.Curr Top Microbiol Immunol. 1996; 211: 201-210PubMed Google Scholar, 20Ru M Shustik C Rassart E Graffi murine leukemia virus: molecular cloning and characterization of the myeloid leukemia-inducing agent.J Virol. 1993; 67: 4722-4731PubMed Google Scholar The Graffi-1.4 U3 region contains a TATA and CAAT box and multiple cis-acting regions that play a major role in viral cell specificity. Several hematopoietic transcription factors bind to Graffi-1.4 LTR sequences, including members of the GATA family of transcription factors GATA1, GATA2, and GATA3, and core binding factor family members-αβ.8Barat C Rassart E Members of the GATA family of transcription factors bind to the U3 region of Cas-Br-E and graffi retroviruses and transactivate their expression.J Virol. 1998; 72: 5579-5588PubMed Google Scholar, 9Barat C Rassart E Nuclear factors that bind to the U3 region of two murine myeloid leukemia-inducing retroviruses, Cas-Br-E and Graffi.Virology. 1998; 252: 82-95Crossref PubMed Scopus (6) Google Scholar Rearrangements in known oncogenes such as c-myc, Fli-1, and Spi-1 are relatively rare in tumors induced by Graffi-1.4, indicating that other genes are involved in these myeloid leukemias.21Denicourt C Edouard E Rassart E Oncogene activation in myeloid leukemias by Graffi murine leukemia virus proviral integration.J Virol. 1999; 73: 4439-4442PubMed Google Scholar Recently, 79 Graffi-1.4-tagged disease genes were identified in common integrations sites in myeloid tumors, of which the majority were not found in lymphomas and monocytic leukemias in other screens, suggesting a more specific involvement in AML.22Erkeland SJ et al.Large-scale identification of disease genes involved in acute myeloid leukemia.J Virol. 2004; 78: 1971-1980Crossref PubMed Scopus (35) Google Scholar Expression of proto-oncogenes can be activated or increased by a retrovirus by several mechanisms (Figure 1). First, when the provirus integrates in the promoter region 5′ of a target gene in the forward orientation, it may activate the gene through promoter sequences in the LTR (Figure 1a, upper panel). Well-documented examples of this mechanism are the ecotropic virus integration site (VIS) 1 (Evi1),23Copeland NG Jenkins NA Retroviral integration in murine myeloid tumors to identify Evi-1, a novel locus encoding a zinc-finger protein.Adv Cancer Res. 1990; 54: 141-157Crossref PubMed Scopus (11) Google Scholar, 24Mucenski ML Taylor BA Copeland NG Jenkins NA Chromosomal location of Evi-1, a common site of ecotropic viral integration in AKXD murine myeloid tumors.Oncogene Res. 1988; 2: 219-233PubMed Google Scholar, 25Mucenski ML et al.Identification of a common ecotropic viral integration site, Evi-1, in the DNA of AKXD murine myeloid tumors.Mol Cell Biol. 1988; 8: 301-308Crossref PubMed Scopus (179) Google Scholar Friend leukemia integration-1 (Fli-1),26Ben-David Y Giddens EB Bernstein A Identification and mapping of a common proviral integration site Fli-1 in erythroleukemia cells induced by Friend murine leukemia virus.Proc Natl Acad Sci USA. 1990; 87: 1332-1336Crossref PubMed Scopus (171) Google Scholar c-myc,27Selten G Cuypers HT Zijlstra M Melief C Berns A Involvement of c-myc in MuLV-induced T cell lymphomas in mice: frequency and mechanisms of activation.EMBO J. 1984; 3: 3215-3222PubMed Google Scholar, 28Corcoran LM Adams JM Dunn AR Cory S Murine T lymphomas in which the cellular myc oncogene has been activated by retroviral insertion.Cell. 1984; 37: 113-122Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 29Cory S et al.Activation of the c-myc oncogene in B and T lymphoid tumors.Curr Top Microbiol Immunol. 1984; 113: 161-165PubMed Google Scholar and c-myb.30Rosson D Dugan D Reddy EP Aberrant splicing events that are induced by proviral integration: implications for myb oncogene activation.Proc Natl Acad Sci USA. 1987; 84: 3171-3175Crossref PubMed Scopus (38) Google Scholar, 31Rosson D Reddy EP Mechanism of activation of the myb oncogene in myeloid leukemias.Ann NY Acad Sci. 1987; 511: 219-231Crossref PubMed Scopus (4) Google Scholar, 32Mukhopadhyaya R Wolff L New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation.J Virol. 1992; 66: 6035-6044PubMed Google Scholar This may also result in the overexpression of aberrant splice variants (Figure 1a, lower panel). A recently published example is the expression of the ∼150-kDa Gnn isoform (Gnn107) in the NFS107 cell line owing to viral insertion in the Evi12 locus.33van den Akker E Vankan-Berkhoudt Y Valk PJ Lowenberg B Delwel R The common viral insertion site Evi12 is located in the 5′-noncoding region of Gnn, a novel gene with enhanced expression in two subclasses of human acute myeloid leukemia.J Virol. 2005; 79: 5249-5258Crossref PubMed Scopus (5) Google Scholar Second, when the provirus integrates near a target gene, it may increase expression by the endogenous promoter-enhancing activity of viral LTR sequences (Figure 1b upper panel)1Jonkers J Berns A Retroviral insertional mutagenesis as a strategy to identify cancer genes.Biochim Biophys Acta. 1996; 1287: 29-57PubMed Google Scholar, 6Wolff L Contribution of oncogenes and tumor suppressor genes to myeloid leukemia.Biochim Biophys Acta. 1997; 1332: F67-F104PubMed Google Scholar or by enhancing the stability of the messenger RNA (Figure 1b lower panel) when the virus integrates in the 3′ untranslated region. The latter mechanism has been described for the commonly targeted Sox4 gene.34Boyd KE et al.Sox4 cooperates with Evi1 in AKXD-23 myeloid tumors via transactivation of proviral LTR.Blood. 2006; 107: 733-741Crossref PubMed Scopus (39) Google Scholar Viral integration may also inactivate a gene owing to disruption of the gene structure (Figure 1c). For instance, the tumor suppressor genes p5335Ben David Y Prideaux VR Chow V Benchimol S Bernstein A Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus.Oncogene. 1988; 3: 179-185PubMed Google Scholar, 36Ben-David Y Lavigueur A Cheong GY Bernstein A Insertional inactivation of the p53 gene during friend leukemia: a new strategy for identifying tumor suppressor genes.New Biol. 1990; 2: 1015-1023PubMed Google Scholar and neurofibromatosis type 1 (Nf1) are affected in this way. However, this occurs less frequently than the activation of proto-oncogenes.37Largaespada Jr, DA Shaughnessy JD Jenkins NA Copeland NG Retroviral integration at the Evi-2 locus in BXH-2 myeloid leukemia cell lines disrupts Nf1 expression without changes in steady-state Ras-GTP levels.J Virol. 1995; 69: 5095-5102PubMed Google Scholar, 38Buchberg AM Bedigian HG Jenkins NA Copeland NG Evi-2, a common integration site involved in murine myeloid leukemogenesis.Mol Cell Biol. 1990; 10: 4658-4666Crossref PubMed Scopus (88) Google Scholar As will be discussed later in this review, more efficient models to detect tumor suppressor genes in retroviral screens have been or are being developed. In some cases, intragenic integrations will result in the expression of truncated protein with altered function. For example, viral integrations in c-myb result in either carboxyl C- or amino N-terminal inactivating truncations.6Wolff L Contribution of oncogenes and tumor suppressor genes to myeloid leukemia.Biochim Biophys Acta. 1997; 1332: F67-F104PubMed Google Scholar, 30Rosson D Dugan D Reddy EP Aberrant splicing events that are induced by proviral integration: implications for myb oncogene activation.Proc Natl Acad Sci USA. 1987; 84: 3171-3175Crossref PubMed Scopus (38) Google Scholar The Notch1 gene forms another interesting example because virus integration results in the overexpression of the cytoplasmic active form of the Notch-1 receptor.39Girard L et al.Frequent provirus insertional mutagenesis of Notch1 in thymomas of MMTVD/myc transgenic mice suggests a collaboration of c-myc and Notch1 for oncogenesis.Genes Dev. 1996; 10: 1930-1944Crossref PubMed Scopus (193) Google Scholar, 40Lee JS Ishimoto A Honjo T Yanagawa S Murine leukemia provirus-mediated activation of the Notch1 gene leads to induction of HES-1 in a mouse T lymphoma cell line, DL-3.FEBS Lett. 1999; 455: 276-280Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 41Yanagawa S et al.Identification of Notch1 as a frequent target for provirus insertional mutagenesis in T-cell lymphomas induced by leukemogenic mutants of mouse mammary tumor virus.J Virol. 2000; 74: 9786-9791Crossref PubMed Scopus (32) Google Scholar Genes in the proximity of a VIS are usually being referred to as VIS genes. A VIS gene identified in multiple independent tumors is termed a common integration site (CIS) gene. When two or more CIS genes are found in a single tumor, it may be considered suggestive that these genes cooperate in collaborating oncogenic pathways. However, this interpretation should be made with caution. For instance, leukemia viruses frequently target the proto-oncogene Sox4 in the 3′ untranslated region in tumors also harboring integrations that activate Evi1 expression. Although this could be suggestive of a collaboration between transcriptional activities of the Sox4 and Evi1 proteins, it appears that Sox4 acts as a strong transactivator of proviral LTRs, leading to increased Evi1 expression.34Boyd KE et al.Sox4 cooperates with Evi1 in AKXD-23 myeloid tumors via transactivation of proviral LTR.Blood. 2006; 107: 733-741Crossref PubMed Scopus (39) Google Scholar In this example, virus integration in Sox4 is thus not cooperative in the sense that a synergizing oncogenic pathway is activated, but it enhances the transforming effects of Evi1 by upregulating Evi1 expression.34Boyd KE et al.Sox4 cooperates with Evi1 in AKXD-23 myeloid tumors via transactivation of proviral LTR.Blood. 2006; 107: 733-741Crossref PubMed Scopus (39) Google Scholar Because tumors caused by replication-competent retroviruses are oligoclonal, it is difficult to establish collaboration between oncogenes in individual tumors.42Largaespada DA Genetic heterogeneity in acute myeloid leukemia: maximizing information flow from MuLV mutagenesis studies.Leukemia. 2000; 14: 1174-1184Crossref PubMed Scopus (28) Google Scholar A more direct approach to identify cooperating disease genes is to perform retroviral screens in mouse strains with a known genetic defect predisposing to cancer.43Mikkers H Berns A Retroviral insertional mutagenesis: tagging cancer pathways.Adv Cancer Res. 2003; 88: 53-99Crossref PubMed Google Scholar In these screens, oncogene cooperation is accompanied by an earlier onset of disease in mutant mice when compared with wild-type littermates. In this way, genes collaborating with the loss of p27Kip1 and Cdkn2a tumor suppressor genes have been identified.44Hwang HC et al.Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis.Proc Natl Acad Sci USA. 2002; 99: 11293-11298Crossref PubMed Scopus (99) Google Scholar, 45Lund AH et al.Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.Nat Genet. 2002; 32: 160-165Crossref PubMed Scopus (195) Google Scholar This strategy can be further improved by using virus strains with reduced replication abilities. For instance, using the cloned amphotropic virus 4070A in an AML inv(16) mouse model, the genes Plag1 and Plagl2 cooperating with the oncogenic fusion protein core binding factor-β-MYH11 have been identified.46Castilla LH et al.Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia.Proc Natl Acad Sci USA. 2004; 101: 4924-4929Crossref PubMed Scopus (112) Google Scholar, 47Landrette SF et al.Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11.Blood. 2005; 105: 2900-2907Crossref PubMed Scopus (101) Google Scholar In a number of recent studies, hematopoietic stem cells and progenitor cells have been infected with replication-defective viruses in vitro, which were then either maintained in culture or transplanted in recipient mice to select transforming events and to identify the genes involved therein. This has proved to be a good alternative for in vivo infection with replication-competent virus strains. For instance, it was shown that integrations of a replication-defective virus containing a marker gene caused clonal dominance of nonmalignant stem cells when integrations occurred 5′ of the Evi1 gene, suggesting that deregulation of Evi1 expression results in clonal expansion of (pre-)malignant cells.48Kustikova O et al.Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.Science. 2005; 308: 1171-1174Crossref PubMed Scopus (304) Google Scholar This result was confirmed in a study showing that ectopic Evi1 overexpression sufficed to immortalize immature myeloid progenitors, without the requirement of other cooperating events.49Du Y Jenkins NA Copeland NG Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells.Blood. 2005; 106: 3932-3939Crossref PubMed Scopus (161) Google Scholar An additional advantage of this strategy is that cell populations transformed in this way are often clonal, which simplifies the identification of tumor-initiating and -cooperating events, particularly when replication-defective viral vectors are used that already contain an oncogene (Sox4) or a multidrug resistance gene (Mdr1) as the first "hit".50Du Y Spence SE Jenkins NA Copeland NG Cooperating cancer-gene identification through oncogenic-retrovirus-induced insertional mutagenesis.Blood. 2005; 106: 2498-2505Crossref PubMed Scopus (109) Google Scholar, 51Modlich U et al.Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.Blood. 2005; 105: 4235-4246Crossref PubMed Scopus (159) Google Scholar This approach has, e.g., unveiled cooperativity between transcription factor Sox4 and Mefc2, a basic helix-loop-helix transcription factor.49Du Y Jenkins NA Copeland NG Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells.Blood. 2005; 106: 3932-3939Crossref PubMed Scopus (161) Google Scholar However, a quick survey of the genes hit by the Sox4 or Mdr1 -containing vectors that are reported in these studies reveals that they do not converge into one or a limited number of gene classes, regulatory pathways or networks, which would have been predicted in the case of strong gene cooperativity. This unexpected heterogeneity might indicate that genes located near vector integrations may not always be the ones that cooperate, but that more distant genes are involved. It is even conceivable that a more complex scenario applies, e.g., in which the transcription regulatory complexes recruited to the viral LTR affect the expression of genes that are far away from the virus integration in a linear way, but, nevertheless, in close spatial proximity owing to conformational properties of the chromatin. Notably, this would also have important ramifications for elucidation of the cooperating events involved in leukemic transformation of hematopoietic cells in retrovirus-based gene therapy protocols. In any case, it is essential that candidate cooperating genes identified in this type of screens are functionally tested for their collaborative action, as was carried out in the example of Sox4 and Mefc2.49Du Y Jenkins NA Copeland NG Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells.Blood. 2005; 106: 3932-3939Crossref PubMed Scopus (161) Google Scholar Retroviral insertion mutagenesis has been successfully applied to screen for oncogenes involved in different types of cancer, including several hematological neoplasms,1Jonkers J Berns A Retroviral insertional mutagenesis as a strategy to identify cancer genes.Biochim Biophys Acta. 1996; 1287: 29-57PubMed Google Scholar, 6Wolff L Contribution of oncogenes and tumor suppressor genes to myeloid leukemia.Biochim Biophys Acta. 1997; 1332: F67-F104PubMed Google Scholar mammary carcinomas,52van Leeuwen F Nusse R Oncogene activation and oncogene cooperation in MMTV-induced mouse mammary cancer.Semin Cancer Biol. 1995; 6: 127-133Crossref PubMed Scopus (47) Google Scholar, 53Callahan R MMTV-induced mutations in mouse mammary tumors: their potential relevance to human breast cancer.Breast Cancer Res Treat. 1996; 39: 33-44Crossref PubMed Scopus (60) Google Scholar and brain tumors.54Johansson FK et al.Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging.Proc Natl Acad Sci USA. 2004; 101: 11334-11337Crossref PubMed Scopus (103) Google Scholar The availability of inverse polymerase chain reaction, vectorette- and splinkerette-polymerase chain reaction strategies,55Birney E et al.Ensembl 2006.Nucleic Acids Res. 2006; 34: D556-D561Crossref PubMed Scopus (331) Google Scholar and genome database programs like Ensembl56Hui EK Wang PC Lo SJ PCR-based strategies to clone unknown DNA regions from known foreign integrants. An overview.Methods Mol Biol. 2002; 192: 249-274PubMed Google Scholar have greatly increased the efficiency of this strategy.18Kim R et al.Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas.J Virol. 2003; 77: 2056-2062Crossref PubMed Scopus (93) Google Scholar, 45Lund AH et al.Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.Nat Genet. 2002; 32: 160-165Crossref PubMed Scopus (195) Google Scholar, 57Mikkers H et al.High-throughput retroviral tagging to identify components of specific signaling pathways in cancer.Nat Genet. 2002; 32: 153-159Crossref PubMed Scopus (325) Google Scholar, 58Suzuki T et al.New genes involved in cancer identified by retroviral tagging.Nat Genet. 2002; 32: 166-174Crossref PubMed Scopus (362) Google Scholar, 59Joosten M et al.Large-scale identification of novel potential disease loci in mouse leukemia applying an improved strategy for cloning common virus integration sites.Oncogene. 2002; 21: 7247-7255Crossref PubMed Scopus (38) Google Scholar, 60Valk PJ Joosten M Vankan Y Lowenberg B Delwel R A rapid RT-PCR based method to isolate complementary DNA fragments flanking retrovirus integration sites.Nucleic Acids Res. 1997; 25: 4419-4421Crossref PubMed Scopus (15) Google Scholar, 61Li J et al.Leukaemia disease genes: large-scale cloning and pathway predictions.Nat Genet. 1999; 23: 348-353Crossref PubMed Scopus (206) Google Scholar, 62Uren AG Kool J Berns A van Lohuizen M Retroviral insertional mutagenesis: past, present and future.Oncogene. 2005; 24: 7656-7672Crossref PubMed Scopus (218) Google Scholar Notwithstanding these improvements, an often-discussed limitation of retroviral screens is its low power to identify tumor suppressor genes. It has recently been shown that MLV does not integrate fully randomly in the genome, but has a bias for insertion near the start of transcriptional units of actively transcribed genes, thus leaving the gene structure intact.63Wu X Li Y Crise B Burgess SM Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Crossref PubMed Scopus (1144) Google Scholar Classically, according to Knudson's two-hit model, identification of tumor suppressor genes would require disruption of both alleles, which predictably occurs at much lower frequency than single allele hits.64Knudson Jr, AG Mutation and cancer: statistical study of retinoblastoma.Proc Natl Acad Sci USA. 1971; 68: 820-823Crossref PubMed Scopus (5622) Google Scholar On the other hand, there is growing evidence that haploinsufficiency, i.e., inactivation of a single allele, may also result in the reduction of tumor suppressor protein levels sufficient for contribution to tumorigenesis.65Hohenstein P Tumour suppressor genes—one hit can be enough.PLoS Biol. 2004; 2: E40Crossref PubMed Scopus (13) Google Scholar, 66Santarosa M Ashworth A Haploinsufficiency for tumour suppressor genes: when you don't need to go all the way.Biochim Biophys Acta. 2004; 1654: 105-122Crossref PubMed Scopus (166) Google Scholar Nonetheless, tumor suppressor genes are clearly underrepresented in currently performed retroviral screens. Moreover, intragenic viral insertions may not always affect potential tumor suppressor genes, but could cause enhanced expression of a more distant gene with oncogenic properties. This complication could be avoided by the use of promoter-less or s