Controlled Gene Expression with a Reverse Tetracycline-Regulated Retroviral Vector (RTRV) System

Watsuji, Toshikazu; Okamoto, Yoshichika; Emi, Nobuhiko; Katsuoka, Yoji; Hagiwara, Masatoshi

doi:10.1006/bbrc.1997.6705

Cited by 57 publications

(46 citation statements)

References 15 publications

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“…Full length CL100 cDNA was cloned into a retroviral vector (pLRT) containing the two components of the reverse tetracycline-regulated (rtTA) system as described (Watsuji et al, 1997). UCI101 and A2780 cells were infected with the retrovirus pLRT-CL100 essentially as described (Watsuji et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

“…UCI101 and A2780 cells were infected with the retrovirus pLRT-CL100 essentially as described (Watsuji et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

“…To analyse the biological e ects of CL100 in more detail and eliminate potential problems from clonal variation, we generated three UCI101 clones with conditional expression of CL100 (U8 and U28) using an amphotrophic retrovirus (Watsuji et al, 1997) expressing CL100 under the control of a tetracycline regulated promoter (rtTA, tet on system). The addition of doxycycline (1 mg/ml) to the culture medium induced expression of CL100 after 48 h (20-fold increased) (Figure 5a), producing CL100 expression levels comparable to those found in IOSE cells.…”

Section: Cl100 Expression Decreases Lamellipodia Filopodia and Cellumentioning

confidence: 99%

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CL100 expression is down-regulated in advanced epithelial ovarian cancer and its re-expression decreases its malignant potential

et al. 2002

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Although early stage ovarian cancer can be e ectively treated with surgery and chemotherapy, the majority of cases present with advanced disease, which remains essentially incurable. Unfortunately, little is known about the genes important for the development and progression of this disease. In this study, the expression of 68 phosphatases was determined in immortalized ovarian epithelial cells (IOSE) and compared to ovarian cancer cell lines. CL100, a dual speci®city phosphatase, displayed 10 ± 25-fold higher expression in normal compared to malignant ovarian cell lines. Immunohistochemical staining of normal ovaries and 68 ovarian cancer specimens con®rmed this di erential expression. Re-expression of CL100 in ovarian cancer cells decreased adherent and nonadherent cell growth and induced phenotypic changes including loss of ®lopodia and lamellipodia with an associated decrease in cell motility. Induced expression of CL100 in ovarian cancer cells suppressed intraperitoneal tumor growth in nude mice. These results show for the ®rst time that CL100 expression is altered in human ovarian cancer, that CL100 expression changes cell morphology and motility, and that it suppresses intraperitoneal growth of human ovarian epithelial cancer. These data suggest that down-regulation of CL100 may play a role in the progression of human ovarian cancer.

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Section: Methodsmentioning

confidence: 99%

“…UCI101 and A2780 cells were infected with the retrovirus pLRT-CL100 essentially as described (Watsuji et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

Section: Cl100 Expression Decreases Lamellipodia Filopodia and Cellumentioning

confidence: 99%

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CL100 expression is down-regulated in advanced epithelial ovarian cancer and its re-expression decreases its malignant potential

et al. 2002

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“…[11][12][13][14][15][16] Another strategy is to combine both components into one self-regulating virus so that target cells only need to be infected by one virus to allow regulatable expression. 7,[17][18][19][20][21][22][23][24][25][26] Recombinant adeno-associated viral (rAAV) vectors have several properties that make them one of the most promising vehicles for gene delivery to the CNS. 27 These vectors have been reported to infect and transduce both dividing and nondividing cells, including neurons, with minimal cellular toxicity or host immune response.…”

Section: Introductionmentioning

confidence: 99%

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

et al. 2004

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Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

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“…Mutation of the tTA produced the reverse tetracycline transactivator (rtTA) which induces gene expression in the presence of doxycycline (Dox). 7 Modifications of the 'on' system include the construction of a self-contained single bacterial plasmid, 8 a retroviral vector 9 and an autoregulatory bicistronic plas-mid 10 all of which were reported during the course of our research. In this study we compare the regulatory response of the 'on system' when the components are combined in self-contained retroviral or plasmid vectors in a standard or autoregulatory format.…”

Section: Introductionmentioning

confidence: 97%

A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications

et al. 2000

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The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule, truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids

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Controlled Gene Expression with a Reverse Tetracycline-Regulated Retroviral Vector (RTRV) System

Cited by 57 publications

References 15 publications

CL100 expression is down-regulated in advanced epithelial ovarian cancer and its re-expression decreases its malignant potential

CL100 expression is down-regulated in advanced epithelial ovarian cancer and its re-expression decreases its malignant potential

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications

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