Powerful and versatile enhancer-promoter unit for mammalian expression vectors

Foecking, Mary K.; Hofstetter, H.

doi:10.1016/0378-1119(86)90137-x

Cited by 256 publications

(129 citation statements)

References 5 publications

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“…Surprisingly, the ratio of CAT to LacZ was severalfold lower for vLZCC than for pLZCC. This difference may reflect an effect of the integrative state of the provirus, although this was not observed in mammalian cells (10). For the other six vectors, however, the relative expression of the two genes was not greatly affected by the state of integration.…”

Section: Resultsmentioning

confidence: 86%

“…The relative levels of CAT expression were roughly twofold higher when the cat gene was placed directly at the 3' end of the EMCV IRES (pLZIC2 and -3) than when it was separated by a 29-bp untranslated fragment (pLZIC1) or expressed from the RSV promoter (pLZUC). The highest levels of CAT expression were obtained from the CMV promoter, which was previously shown to be more powerful than the RSV promoter in mammalian expression vectors (10 spliced message was only 5 to 10% that of the IRES vectors. There was no CAT expression from the 232-bp EMCV fragment of pLZAIC, which confirms the requirement of an IRES for internal initiation.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

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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“…11,12 To study the role of CD46 in Ad35 vector-mediated transduction into human bone marrow CD34 + cells, we evaluated the relationship between CD46 expression levels and the transduction efficiencies of Ad35 vector containing the CMV promoter. The CMV promoter is generally regarded as one of the most powerful Optimization of promoter in Ad35 vectors into human hematopoietic progenitors F Sakurai et al promoters 24 and is widely used in transduction experiments. As shown in Figure 2a, almost all the bone marrow CD34 + cells expressed high levels of CD46, similar to the cord blood CD34 + cells.…”

Section: Construction Of E1/e3-deleted Ad35 Vectors By the Improved Imentioning

confidence: 99%

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

et al. 2005

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Adenoviral gene transfer to hematopoietic stem cells (HSCs)/ progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34 + cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34 + cells and primitive CD34 + subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1a promoter (EF1a promoter), the human cytomegalovirus (CMV) immediateearly 1 gene enhancer/b-actin promoter with b-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34 + cells and the immature subsets (CD34 + CD38 low/À and CD34 + AC133 + subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34 + cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

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“…CMV promoter is the most commonly used promoter in mammalian expression plasmids (Boshart et al, 1985;Foecking and Hofstetter, 1986;Hundt et al, 2009) but has been shown to be active in a broad range of cell types as has simian virus 40 (Robbins and Ghivizzani, 1998;Kimchi-Sarfaty et al, 2003;Park et al, 2004). RSV long terminal repeat, which is activated by the growth factor ligands of tyrosine kinase receptors (Cosgaya et al, 1997), is often used to enhance gene expression (Machon et al, 1998;Majhen et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Transcriptional activity of an ovarian-specific promoter from rat in dairy goat granulosa cells

Liu

Yang²,

Cui³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Ovarian-specific promoter 1 (OSP-1) is a retrovirus-like element isolated from the complementary DNA library of rat that has been thought to be specifically expressed in ovary. To exploit this promoter in dairy goat ovary granulosa cells (GCs), OSP-1 from rat was used to construct the reporter vector pOSP-1-EGFP, in which egfp coding for enhanced green fluorescent protein (EGFP) was used as a reporter to examine the activity of OSP-1 in GCs. EGFP was successfully expressed in dairy goat GCs transfected with pOSP-1-EGFP. Reverse transcriptasepolymerase chain reaction analysis confirmed the tissue-specific transcription of EGFP messenger RNA in dairy goat GCs transfected with pOSP-1-EGFP. We concluded that OSP-1 promoter from rat can specifically drive foreign gene expression in dairy goat GCs. Thus, we obtained a tissue-specific regulation element and provided a potential tool for the research of regulation and development of the ovary in dairy goats.

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Powerful and versatile enhancer-promoter unit for mammalian expression vectors

Cited by 256 publications

References 5 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.

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

Transcriptional activity of an ovarian-specific promoter from rat in dairy goat granulosa cells

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