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

Overell, Robert W.; Weisser, K E; Cosman, D

doi:10.1128/mcb.8.4.1803

Cited by 36 publications

(16 citation statements)

References 24 publications

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“…Therefore, we used dual-promoter lentiviral vectors. Although there were several studies reporting that dual-promoter vectors based on oncoretrovirus backbones showed severe reduction of transgene expression due to promoter interference (Emerman and Temin, 1984;Overell et al, 1988), a similar strategy was successful when using lentiviral vectors (Yu et al, 2003). Thus, Yu and colleagues have achieved efficient and consistent coexpression of two genes in cord-blood CD34+ HSCs or primary endothelial cells (Yu et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

Hui

Mostoslavsky

Eruslanov

et al. 2008

Journal of Immunological Methods

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In-depth studies of innate immunity require efficient genetic manipulation of macrophages, which is especially difficult in primary macrophages. We have developed a lentiviral system for inducible gene expression both in macrophage cell lines and in primary macrophages. A transgenic mouse strain C3H.TgN(SRA-rtTA) that expresses reverse tetracycline transactivator (rtTA) under the control of macrophage-specific promoter, a modified human scavenger receptor A (SR-A) promoter was generated. For gene delivery, we constructed a dual-promoter lentiviral vector, in which expression of a "gene-of-interest" is driven by a doxycycline-inducible promoter and the expression of a selectable surface marker is driven by an independent constitutive promoter UBC. This vector is used for transduction of bone marrow-derived macrophage precursors. The transduced cells can be enriched to 95-99% purity using marker-specific monoclonal antibodies, expanded and differentiated into mature macrophages or myeloid dendritic cells. We also successfully used this approach for inducible protein expression in hard to transfect macrophage cell lines.Because many proteins, which are expressed by activated or infected macrophages, possess cytotoxic, anti-proliferative or pro-apoptotic activities, generation of stable macrophage cell lines that constitutively express those proteins is impossible. Our method will be especially useful to study immunity-related macrophage proteins in their physiological context during macrophage activation or infection.

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Section: Discussionmentioning

confidence: 99%

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

Hui

Mostoslavsky

Eruslanov

et al. 2008

Journal of Immunological Methods

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“…We also found that the cytoplasmic domain of TACE is not required for any of the shedding events examined and that the cysteine-rich domain of TACE is required for shedding of one of the substrates while affecting the inhibitor sensitivity of another substrate's shedding. (23). The resulting cultures were expanded, and clones were obtained by limiting dilution.…”

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confidence: 99%

Functional Analysis of the Domain Structure of Tumor Necrosis Factor-α Converting Enzyme

Reddy¹,

Slack²,

Davis³

et al. 2000

Journal of Biological Chemistry

479

435

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Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-␣ converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or "shedding" of several cell-surface proteins, including TNF and p75 TNFR. We established a TACE-reconstitution system using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, we identified two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, we studied the function of the different domains of TACE in three shedding activities. We found that TACE must be expressed with its membraneanchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding required the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding required only the tethered TACE catalytic domain.

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“…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.…”

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

Cited by 36 publications

References 24 publications

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

Functional Analysis of the Domain Structure of Tumor Necrosis Factor-α Converting Enzyme

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

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