Lack of retrovirus gene expression in somatic cell hybrids of friend cells and teratocarcinoma cells with a teratocarcinoma phenotype.

Asche, W; Colletta, Giulia; Warnecke, G; Nobis, Peter; Pennie, S; King, R; Ostertag, Wolfram

doi:10.1128/mcb.4.5.923

Cited by 12 publications

(13 citation statements)

References 41 publications

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“…In contrast, the second restriction is poorly characterized, occurs at an unknown time point, and acts in cis to maintain the initial block in transcription. The second block was identified in early studies which showed that differentiation induction or cell fusion relieved the first block to virus infection, as demonstrated by de novo infection, but did not permit expression of provirus that had integrated prior to differentiation (4,19,21,43).…”

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confidence: 99%

Host cis -Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

Laker

Meyer

Schopen

et al. 1998

J Virol

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The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by acis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independentcis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy.

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confidence: 99%

Host cis -Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

Laker

Meyer

Schopen

et al. 1998

J Virol

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“…Immunofluorescence studies were performed as described (26). SSEA-1, a gift of P. Goodfellow, is a mouse hybridoma line secreting antibodies that react specifically with undifferentiated EC cells (32).…”

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confidence: 99%

Retroviral mutants efficiently expressed in embryonal carcinoma cells.

Franz¹,

Hilberg²,

Seliger³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The myeloproliferative sarcoma virus (MPSV) is a unique member of the Moloney murine sarcoma virus family. Due to mutations in the U3 region of its long terminal repeat, MPSV has an expanded host range that includes cells of the hematopoietic compartment. Using a MPSV recombinant containing the gene for neomycin-resistance (NeoR-MPSV), we demonstrate that the host range of MPSV also includes undifferentiated F9 embryonal carcinoma cells. Transfer of G418-resistance with NeORMPSV to F9 cells is almost as efficient as G418-resistance transfer to fibroblasts, in contrast to G418-resistance transfer to PCC4 embryonal carcinoma cells, which is at least 3 orders of magnitude lower.To isolate NeoRMPSV mutants that are efficiently expressed in PCC4 cells, G418-resistant PCC4 cell lines were induced to differentiate, and the provirus was rescued by superinfection with murine leukemia'virus. Viral isolates (PCMV-5 and -6; PCMV = PCC4 cell-passaged Neo".MPSV) were obtained and assayed for expression in embryonal carcinoma cells. The efficiency of NeoR transfer was equally as high in both F9 and PCC4 as in fibroblasts. mos oncogene expression was unaltered as judged by transformation capability. No gross alteration in the coding region and in the long terminal repeat was detectable by restriction enzyme analysis. NeoR-MPSV and its mutants PCMV-5 and -6 can thus be utilized as vectors for the efficient transduction of genes into embryonic cells.

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“…However, long-term expression mediated by integrated proviruses in primary cells has been difficult to achieve. Retroviral regulatory elements are repressed in numerous cell types, including embryonic stem (ES) cells and hematopoietic stem (HS) cells (1,3). For example, vectors that are functional in mature hematopoietic cells are often not expressed in blood cells of animals transplanted with the infected stem cells (18,19,31).…”

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Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

Cherry

Biniszkiewicz

Parijs

et al. 2000

Molecular and Cellular Biology

253

205

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(18,19,31). In particular, the lack of significant provirus transcription in ES cells and their differentiated descendants has hampered the use of retroviral vectors in transgenic experiments (5,12,32). Interestingly, this block in provirus expression is maintained upon differentiation of infected cells despite the fact that primary infection of cells after differentiation results in efficient expression (6,7,26).Transcriptional repression is thought to be mediated by both cis-acting de novo methylation of the integrated proviruses and cell-type-specific trans-acting transcriptional repressors (5, 9, 23). The effect of trans-acting factors on retroviral expression through binding of specific sequences within the promotors of retroviruses has been examined in many studies (29,30,35). In fact, the mouse stem cell virus (MSCV) long terminal repeat (LTR) was generated by the modification of the sequences within the LTR to increase the affinity for positive factors and decrease the affinity for negative regulators (20).In contrast, the role of methylation in silencing has been less clear. DNA methylation is thought to be a general mechanism used by cells to silence foreign DNA and may be involved in the cell defense against transposable elements (39). DNA methylation has also been associated with the repression of gene expression and the silencing of viral control elements (2,14,38). Exogenously introduced retroviruses silenced in vitro and in vivo can be reactivated by treatments that result in genomewide demethylation. In addition, transcriptionally silent endogenous retroviral elements are reactivated upon loss of genomic methylation in Dnmt1 knockout mice (38). Therefore, DNA methylation is thought to causally repress expression of retroviral promoters in a variety of cell types.ES cells provide a good model to study the role of DNA methylation in retroviral silencing. First, it was demonstrated that ES cells have high de novo methylation activity, which leads to effective methylation of integrated retroviral vectors, while little or no de novo methylation activity was detected in differentiated cells (21). In addition, ES cells were genetically modified to alter the endogenous level of DNA methylation by the targeted disruption of the maintenance methyltransferase gene Dnmt1. ES cells homozygous for this mutation proliferate normally with their genomic DNA highly demethylated, while differentiated cells and mice die due to the loss of genomic methylation (21,22). Therefore, these modified ES cells are useful to study the effect of DNA methylation on retroviral gene expression. In addition, ES cells can be induced to differentiate in vitro or in vivo, allowing the study of DNA methylation and its effect on long-term expression.Both Moloney virus-based and MSCV-based retroviral vectors have been used for gene transduction in a variety of cells. The MSCV vector is different from the typical Moloney virus vector in that the mutations in the LTR have allowed expression in a larger host range (8,20). To this end, we ...

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Lack of retrovirus gene expression in somatic cell hybrids of friend cells and teratocarcinoma cells with a teratocarcinoma phenotype.

Cited by 12 publications

References 41 publications

Host cis -Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

Host cis -Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

Retroviral mutants efficiently expressed in embryonal carcinoma cells.

Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

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