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NRAS is a proto-oncogene involved in numerous myeloid malignancies. Here, we report on a mouse line bearing a single retroviral long terminal repeat inserted into Nras. This genetic modification resulted in an increased level of wild type Nras mRNA giving the possibility of studying the function and activation of wild type NRAS. Flow cytometry was used to show a variable but significant increase of immature myeloid cells in spleen and thymus, and of T- cells in the spleen. At an age of one week, homozygous mice began to retard compared to their wild type and heterozygous littermates. Two weeks after birth, animals started to progressively lose weight and die before weaning. Heterozygous mice showed a moderate increase of T-cells and granulocytes but survived to adulthood and were fertile. In homozygous and heterozygous mice Gfi1 and Gcsf mRNA levels were upregulated, possibly explaining the increment in immature myeloid cells detected in these mice. The short latency period indicates that Nras overexpression alone is sufficient to cause dose-dependent granulocytosis and Tcell expansion. [ABSTRACT FROM AUTHOR]

Background: Insertional mutagenesis screens in the mouse are an acknowledged approach to identify genes involved in the pathogenesis of cancer. The potential of these screens to identify genes causally involved in tumorigenesis is not only limited to the murine host, but many of these genes have also been proven to be involved in the oncogenic process in man. Results: Through an insertional mutagenesis screen applying murine leukemia viruses in mouse, we found that Cd74 was targeted by proviral insertion in tumors of B-cell origin. This locus encodes a protein playing crucial roles in antigen presentation and B-cell homeostasis, and its deregulation is often associated with cancer in man. The distribution of insertions within the Cd74 locus prompted the identification of an alternative transcript initiated in intron 1 of Cd74 encoding an N-terminally truncated Cd74 isoform in tissues from un-infected mice, and transcriptional activation assays revealed a positive effect on the novel intronic promoter by a formerly described intronic enhancer in the Cd74 locus. Furthermore, we show that the new Cd74 isoform is IFNγ inducible and that its expression is differentially regulated from the canonical Cd74 isoform at the transcriptional level. Conclusions: We here identify Cd74 as a common insertion site in murine B-lymphomas and describe a novel IFNγ-inducible murine Cd74 isoform differentially regulated from the canonical isoform and expressed under the control of an intronic promoter. The distribution and orientation of proviral insertion sites within the Cd74 locus underscores the causal involvement of the isoforms in the murine B-lymphomagenic process. [ABSTRACT FROM AUTHOR]

Abstract: In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI-inbred strain were inoculated with murine leukemia virus mutants. The resulting tumors were analyzed by integration analysis and global gene expression profiling to determine the effect of the retroviral integrations on the nearby genes, and the deregulated pathways in the tumors. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance and origin of recognition pathway as well as downregulation in negative regulators of G1/S transition, indicating increased S-phase initiation in murine T-LBLs. [Copyright &y& Elsevier]

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 × 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells. [ABSTRACT FROM AUTHOR]

Abstract: Neurogranin (Nrgn) is a highly expressed brain-specific protein, which sequesters calmodulin at low Ca2+-levels. We report here on retroviral activation of the Nrgn gene in tumors induced by the T-cell lymphomagenic SL3-3 murine leukemia virus. We have performed a systematic expression analysis of Nrgn in various mouse tissues and SL3-3 induced T-cell tumors. This demonstrated that insertional activation of Nrgn increased RNA and protein expression levels to that observed in brain. Furthermore, elevated Nrgn expression was also observed in some T-cell tumors with no detected provirus integrations into this genomic region. The presented data demonstrate that Nrgn can be produced at high levels outside the brain, and suggest a novel oncogenic role in T-cell lymphomas in mice. [Copyright &y& Elsevier]

SL3-3 murine leukemia virus is a potent inducer of T-lymphomas in mice. Using inbred NMRI mice, it was previously reported that a mutant of SL3-3 with all enhancer Runx (AML1/core) sites disrupted by 3-bp mutations (SL3-3dm) induces predominantly non-T-cell tumors with severely extended latency (S. Ethelberg, J. Lovmand, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:7273-7280, 1997). By use of three-color flow cytometry and molecular and histopathological analyses, we have now performed a detailed phenotypic characterization of SL3-3- and SL3-3dm-induced tumors in this mouse strain. All wild-type induced tumors had clonal T-cell receptor β rearrangements, and the vast majority were CD3+ CD4+ CD8- T-lymphomas. Such a consistent phenotypic pattern is unusual for murine leukemia virus-induced T-lymphomas. The mutant virus induced malignancies of four distinct hematopoietic lineages: myeloid, T lymphoid, B lymphoid, and erythroid. The most common disease was myeloid leukemia with maturation. Thus, mutation of all Runx motifs in the enhancer of SL3-3 severely impedes viral T-lymphomagenicity and thereby discloses a considerable and formerly unappreciated potential of this virus for myeloid leukemia induction. Proviral enhancers with complex structural alterations (deletions, insertions, and/or duplications) were found in most SL3-3dm-induced T-lymphoid tumors and immature myeloid leukemias but not in any cases of myeloid leukemia with maturation, mature B-lymphoma, or erythroleukemia. Altogether, our results indicate that the SL3-3dm enhancer in itself promotes induction of myeloid leukemia with maturation but that structural changes may arise in vivo and redirect viral disease specificity to induction of T-lymphoid or immature myeloid leukemias, which typically develop with moderately shorter latencies. [ABSTRACT FROM AUTHOR]

A murine leukemia virus-derived replication-competent retroviral vector with a translational cassette for the enhanced green fluorescence protein (EGFP) was previously found to function efficiently in cell culture (Jespersen et al., 1999, Gene 239, 227–235). We here report that infection of newborn NIH Swiss mice gives rise to EGFP expression in a majority of spleen cells within the first days after infection. Among the nonadherent spleen cells, B, T, and NK lymphocytes were found to be efficiently marked by EGFP by flow cytometry analysis, whereas the adherent spleen cells were negative in most animals. Analysis at time points up to 60 days after infection reveals a decline in EGFP-positive spleen cells over time. Viremia analysis and PCR analysis of spleen DNA indicate that viruses that have lost the translation cassette predominate at later stages. The results provide a model for efficient gene delivery to spleen cells in a time window of 1 to 2 weeks after infection of newborns. Although this type of vector will not in itself be applicable in the clinic, we envision that efficient in vivo gene delivery will be valuable by analysis of differentiation and proliferation of hematopoietic cells in animal models and by development and evaluation of effectors interfering with these processes. [Copyright &y& Elsevier]