A Multicolor Panel of Novel Lentiviral “Gene Ontology” (LeGO) Vectors for Functional Gene Analysis

Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

doi:10.1038/mt.2008.6

Cited by 305 publications

(230 citation statements)

References 31 publications

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“…Plasmids used were LeGO-iG2, 30 Precision LentiORF Human FOS (Thermo Scientific, OHS5897-202616290), Ras/E1a, 17 pLKO (Sigma), pLKO-p53sh (Sigma), psPax2, pCL-Eco. Viruses were pseudotyped with VSV-G.…”

Section: Packaging Of Retroviral Constructs and Retroviral Transductionmentioning

confidence: 99%

Fos-dependent induction of Chk1 protects osteoblasts from replication stress

et al. 2014

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Section: Packaging Of Retroviral Constructs and Retroviral Transductionmentioning

confidence: 99%

Fos-dependent induction of Chk1 protects osteoblasts from replication stress

et al. 2014

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“…Lentiviral gene ontology (LeGO) vectors (48,49) were used for delivery of adenoviral sequences. PCR-amplified genomic HAdV-5 E1A (GenBank accession no.…”

Section: Primary Cells and Cell Linesmentioning

confidence: 99%

“…Lentiviral particles were produced and titrated according to the method of Weber et al (48). Briefly, particles were pseudotyped with vesicular stomatitis virus G protein (VSV-G).…”

Section: Primary Cells and Cell Linesmentioning

confidence: 99%

“…hMSC or pBRK cells were seeded on 12-well plates (Sarstedt) 12 h before transduction. Adherent cells were transduced at multiplicities of infection (MOIs) of 0.3 particles/cell as described previously (48). Cells were cultivated for 3 to 4 weeks in DMEM-LG (Dulbecco's modified Eagle's medium-low glucose; Gibco) supplemented with 2 mM GlutaMAX (Gibco), 10% preselected FCS (BioWhittaker), 200 g/ml G418 (Calbiochem), and 50 g/ml blasticidin S (InvivoGen).…”

Section: Primary Cells and Cell Linesmentioning

confidence: 99%

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Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes

Speiseder

Hofmann-Sieber

Rodríguez

et al. 2017

J Virol

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Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well.IMPORTANCE It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

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“…Expression of the fusion protein was induced by addition of tetracycline for 24 h at final concentration of 1 mg/ml. LeGO-iG2-Puro C vector 34,35 was a kind gift from Kristoffer Riecken and Boris Fehse. Human SHIP1 cDNA 8 was sub-cloned into the LeGO-iG2-Puro C vector using a BamHI/BamHI strategy.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies

Ehm

Nalaskowski

Wundenberg

et al. 2015

Nucleus

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The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in haematopoietic cells. By converting PI(3,4,5)P 3 to PtdIns(3,4)P 2 at the plasma membrane, SHIP1 modifies PI3-kinase mediated signaling. We have recently demonstrated that SHIP1 is a nucleo-cytoplasmic shuttling protein and SHIP1 nuclear puncta partially colocalize with FLASH, a component of nuclear bodies. In this study, we demonstrate that endogenous SHIP1 localizes to intranucleolar regions of both normal and leukemic haematopoietic cells. In addition, we report that ectopically expressed SHIP1 accumulates in nucleolar cavities and colocalizes with the tumor suppressor protein p53 and components of PML nuclear bodies (e.g. SP100, SUMO-1 and CK2). Moreover, SHIP1 also colocalizes in nucleolar cavities with components of the ubiquitin-proteasome pathway. By using confocal microscopy data, we generated 3D-models revealing the enormous extent of the SHIP1 aggresomes in the nucleolus. Furthermore, treatment of cells with the proteasome inhibitor MG132 causes an enlargement of nucleolar SHIP1 containing structures. Unexpectedly, this accumulation can be partially prevented by treatment with the inhibitor of nuclear protein export Leptomycin B. In recent years, several proteins aggregating in nucleolar cavities were shown to be key factors of neurodegenerative diseases and cancerogenesis. Our findings support current relevance of nuclear localized SHIP1.

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A Multicolor Panel of Novel Lentiviral “Gene Ontology” (LeGO) Vectors for Functional Gene Analysis

Cited by 305 publications

References 31 publications

Fos-dependent induction of Chk1 protects osteoblasts from replication stress

Fos-dependent induction of Chk1 protects osteoblasts from replication stress

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes

The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies

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