The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells

Artelt, Petra; Grannemann, R.; Stocking, Carol; Friel, Jutta; Bartsch, Jörg W.; Hauser, H.

doi:10.1016/0378-1119(91)90134-w

Cited by 132 publications

(60 citation statements)

References 18 publications

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“…We suggest that replacement of 'old-mannered' resistance genes by the novel variants may significantly improve performance of various vectors systems by avoiding deleterious effects of the gene sequence on transgene expression and/or vector titer. 11 In conclusion, we suppose that each of the different novel tools presented here (novel LeGO vectors, drugselectable FPs and codon-optimized antibiotic-resistance genes) may be of great value for researchers working in the fields of cell marking and/or functional gene analysis.…”

Section: Discussionmentioning

confidence: 88%

“…Both, low expression levels and low titers might be related to the length of the respective fusion genes, their primary coding sequences and/or the presence of transcriptional silencers. 11 We therefore in silico analyzed sequences of all resistance markers with regard to human codon usage and the presence of potential aberrant splice sites and/or transcriptional silencers. On the basis of this analysis, we next synthesized codonoptimized versions of the three weakly expressed resistance genes Hygro, Neo and Puro, and used these variants to generate LeGO-G/Neo-opt, LeGO-G/ Hygro-opt and LeGO-G/Puro-opt.…”

Section: Codon-optimized Drug-selectable Markersmentioning

confidence: 99%

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Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis

et al. 2009

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Section: Discussionmentioning

confidence: 88%

Section: Codon-optimized Drug-selectable Markersmentioning

confidence: 99%

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis

et al. 2009

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“…Despite some evidence suggesting that neo r mediates a cis-acting suppression on adjacent promoters, 7,12 reintroduction of neo r into pGC-GFP-loxP * producer cells did not significantly change the expression of GFP. It has not been determined whether the observed effects after the excision of the marker gene cassette are due to promoter suppression and/or changes in the mRNA stability.…”

Section: Figure 7 Facs Analysis Of Gfp Expression In Pa317 Derived Rementioning

confidence: 84%

“…Recently, the expression of neo r has been reported to contribute to transcriptional silencing of adjacent promoters 7,12 and interference with the metabolism of recipient cells. 13,14 In addition, data from clinical gene marking studies using the neo r or a neo r -fusion gene as a selectable marker have shown that immune elimination of circulating genemodified cells may occur after repeated infusions (L Muul and RM Blaese, unpublished observations).…”

Section: Introductionmentioning

confidence: 99%

Generation of a conditionally neor-containing retroviral producer cell line: effects of neor on retroviral titer and transgene expression

et al. 1998

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We have developed a method for generating high-titer required was excised from the provirus by transient Cre retroviral producer cell lines conditionally containing a neoexpression using an adenoviral vector. This strategy led to mycin resistance gene (neo r ) based on the Cre/loxP sysprecise excision of neo r and generation of retroviral supertem. For this, a bicistronic retroviral splicing vector carrying natants containing functional 'neo-less' retroviral particles the green fluorescence protein (GFP) and a marker gene without detrimental effects on the high vector titers found cassette consisting of internal ribosome entry site (IRES) in the parental neo r -containing producer lines. GFP and neo r flanked by loxP sites, was constructed and conexpression was significantly increased after the excision of veniently used to generate a G418 resistant vector proneo r , in both the producer lines and retrovirally transduced ducer cell line. Following titer determination and verification target cells. Reintroduction of neo r did not alter GFP of the biological activity of the retroviral supernatants, the expression, suggesting that the neo r gene and/or its gene selectable expression cassette which was no longer product per se are not acting as a transcriptional silencer.

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“…20 The presence of Neo-R sequences in the MSCV-p210 vectors could lead to reduced BCR-ABL expression when cells are selected in the presence of G418, through a transcriptional silencing mechanism 21 as opposed to the MP-ZEN p210 vector where BCR-ABL is the only gene expressed through the 5Ј LTR. 17 The practical result of the use of the two different BCR-ABL vectors and/or the cell selection system was the obtention of clones with low and high levels of BCR-ABL expression in the pluripotent human cell line UT-7.…”

Section: Figurementioning

confidence: 99%

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7

et al. 2000

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There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors. Leukemia (2000) 14, 662-670.

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The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells

Cited by 132 publications

References 18 publications

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis

Generation of a conditionally neor-containing retroviral producer cell line: effects of neor on retroviral titer and transgene expression

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7

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