Quantitative analysis of gene suppression in integrated retrovirus vectors.

Emerman, Michael; Temin, Howard M.

doi:10.1128/mcb.6.3.792

Cited by 159 publications

(82 citation statements)

References 38 publications

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“…Similarly, in a single experiment, the LacZ titers of pLZSAC and pLZCC were found to be 35 and 40% that of pLZIC1, respectively. These results are consistent with the possibilities that negative interactions between promoters partially inhibit the production of genomic RNA from the LTR (8,9) and that expression of spliced subgenomic mRNA is at the expense of full-length genomic RNA (see below).…”

Section: Resultssupporting

confidence: 79%

“…In most of these tumors, the provirus was found to bear mutations near its 5' end that were correlated with expression of c-myc from the 3' LTR (14). Independently, Emmerman and Temin (8,9) Together, these results provide strong evidence that the EMCV IRES is more efficient than either of the previously used approaches at coexpressing two genes from a recombinant provirus.…”

Section: Resultssupporting

confidence: 49%

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The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

Ghattas¹,

Sanes²,

Majors³

1991

Mol. Cell. Biol.

319

189

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When a retrovirus infects a cell, the viral genome is stably integrated into the host chromosome, efficiently expressed, and faithfully passed to the infected cell's progeny. For these reasons, recombinant retroviral vectors have often been used to express exogenous genes in vertebrate cells (reviewed in references 28, 34, and 52). In some of these cases, it is advantageous to express two exogenous genes from a single proviral genome. In strategies being developed for gene therapy, for example, the retrovirus often contains not only the gene of interest but also a selectable marker. The marker is used to facilitate the isolation of infected cells, which are then used as a source of the potentially therapeutic gene product (reviewed in reference 12).In another set of studies, we and others have used vectors encoding the histochemical marker 3-galactosidase, the product of the Escherichia coli lacZ gene, as lineage tracers in vivo. A single cell is infected by a retrovirus, the proviral genome is inherited by the cell's progeny, and the clonal relatives are identified with the histochemical stain for LacZ (11,13,16,18,45; reviewed in reference 17). To extend this work, we wished to construct an efficient double-expression vector to transfer both lacZ and a second gene to single cells in vivo. If lacZ and a second bioactive gene were reliably coexpressed at high levels, we could use LacZ histochemistry to identify small clones of transgenic cells in a wild-type environment and then seek cell autonomous effects of the second gene by analyzing the number, distribution, and morphology of the labeled cells.In applications such as these, it is essential that the provirus express both genes within the same individual cells. Retroviruses have been successful in evolving strategies for * Corresponding author.coexpressing their own genes. These strategies include synthesis and subsequent processing of fusion proteins, ribosome frameshifting, and regulated splicing to generate subgenomic messages. Unfortunately, achieving balanced expression of multiple exogenous genes from engineered retrovirus vectors has been more problematic. Two approaches have been used previously. The first involves the generation of separate mRNAs by regulated splicing of a single primary transcript expressed from the upstream long terminal repeat (LTR). This strategy mimics that used by retroviruses to generate the env gene product (54). With this approach, expression of one gene is always at the expense of the other, and the ratio of spliced to unspliced mRNA is highly dependent on the context (1,2,48,49). The second approach involves expression of the upstream gene from the retrovirus promoter in the LTR and expression of the downstream gene from an internal promoter. This approach has been used most often in vectors designed for gene therapy (reviewed in references 28 and 34) but is compromised by competitive interference between promoters (4,8,20,39). Thus, individual isolates may express one gene or the other, rather than both.The recent demons...

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Section: Resultssupporting

confidence: 79%

Section: Resultssupporting

confidence: 49%

The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

Ghattas¹,

Sanes²,

Majors³

1991

Mol. Cell. Biol.

319

189

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“…[6][7][8][9][10] The internal promoter-based designs were thought to suffer from the limitation of promoter occlusion where the transcription from one promoter suppresses transcription from another when the two promoters are arranged in the same orientations. [11][12][13] IRESbased designs avoid such problems by expressing multiple genes in one message from a single promoter.…”

mentioning

confidence: 99%

The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site

Byun

Robbins

et al. 1998

Gene Ther

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The internal ribosome entry site (IRES) from the picornaterial neo sequence has a negative effect on expression. virus family has frequently been used to express multiple Analysis of the steady-state RNA levels isolated from genes from a polycistronic message in retroviral vectors.transfected packaging cells showed that the neo-containWhile examining factors affecting levels of gene expression ing retroviral vectors produced significantly lower levels of in IRES-containing retroviral vectors, it was found that RNA than those lacking this bacterial sequence, indicating retroviral vectors expressing the two genes linked by IRES, that neo interferes with expression of the neighboring gene the reporter gene and the selectable marker neo, produced at the level of RNA. Furthermore, the order of genes in the significantly lower levels of protein than those containing a IRES-neo-containing vectors appeared to be more reporter gene alone. This observation has been made with important than in the vector lacking the neo sequence. Our various cDNA sequences. However, when the neo was results suggest that neo has to be used in the retroviral replaced with a different cDNA, the level of gene vector with care, especially when a high level gene expression was increased, often to the level achieved with expression is needed. a vector expressing a single gene, suggesting that the bacKeywords: IRES; neo; retrovirus; gene therapy Retroviral vectors have been the vehicle of choice for many gene transfer experiments and a large number of clinical protocols still employ ex vivo approaches which necessarily involve the use of selectable marker genes to allow easy selection for the successfully transduced cells. 1,2 Of the various dominant selectable markers that are in use for mammalian cells, the neo gene has been the most extensively employed as a marker for retroviral vectors. 3,4 There are two general ways of expressing neo along with the gene of interest. In one type, an independent promoter, either internal or the LTR itself, is used, 5 while in the second type, the neo gene is expressed as part of a multicistronic message using an internal ribosome entry site (IRES). 6-10 The internal promoter-based designs were thought to suffer from the limitation of promoter occlusion where the transcription from one promoter suppresses transcription from another when the two promoters are arranged in the same orientations. 11-13 IRESbased designs avoid such problems by expressing multiple genes in one message from a single promoter.The IRES sequences utilized to date are derived mostly from members of the picornavirus family. They can be isolated from an original viral genome and linked to Correspondence: S Kim, IMBG, Korea Received 26 February 1998; accepted 5 June 1998 unrelated genes to produce polycistronic mRNAs. Indeed, IRES elements have been demonstrated to work in the context of retroviral vectors to express two or more genes. [6][7][8][9][10] Despite the wide use of neo and IRES in retroviral vector designs, factors affecting gen...

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“…This probably reflected, at least in part, the relative transcriptional strengths of the promoters used in murine fibroblasts; however, we were unable to rule out the possibility that some transcriptional interference was occurring. Ultimately, an important determinant of the observed stability of these types of vectors may be whether or not the particular context of the promoter controlling transcription of the selectable marker allows an expression level adequate to confer the selectable phenotype on infected cells (10).…”

mentioning

confidence: 99%

“…In addition, a number of vectors have been constructed in which inserted genes are expressed from heterologous internal promoters. The promoters that have been used to date include those from the simian virus 40 (SV40) early region and the herpes simplex virus thymidine kinase (HSV tk), mouse metallothionein, and rat growth hormone genes (10,20,23,24,35). Internal promoters represent one way in which the gene of interest can be expressed together with a selectable marker in infected cells.…”

mentioning

confidence: 99%

Stably transmitted triple-promoter retroviral vectors and their use in transformation of primary mammalian cells.

Overell¹,

Weisser²,

Cosman³

1988

Mol. Cell. Biol.

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Retroviral vectors were constructed which coexpressed three inserted genes from independent transcriptional promoters in singly infected cells. Several such triple-promoter vectors were constructed with various combinations of oncogenes and selectable drug resistance genes. All expressed three mRNAs of the expected size in infected cells. One vector expressing the v-Ha-ras, v-myc, and neo genes was characterized in detail. This retrovirus did not undergo rearrangement during the process of infection, as judged by Southern analysis, and infection of primary rat embryo fibroblasts demonstrated that ras-myc-cotransformed cells could be selected in G418. This demonstration that retroviral vectors can be used to express three cistrons independently increases their value as gene transfer vehicles, particularly for studies involving oncogene cooperation in primary cells.

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Quantitative analysis of gene suppression in integrated retrovirus vectors.

Cited by 159 publications

References 38 publications

The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site

Stably transmitted triple-promoter retroviral vectors and their use in transformation of primary mammalian cells.

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