The Responsiveness of a Tetracycline-sensitive Expression System Differs in Different Cell Lines

Howe, James R.; Skryabin, Boris V.; Belcher, Scott M.; Zerillo, Cynthia; Schmauss, Claudia

doi:10.1074/jbc.270.23.14168

Cited by 107 publications

(73 citation statements)

References 26 publications

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“…However, a problem with this system is that tet must be present to repress gene expression, and tTA protein is toxic to mammalian cells (Gossen et al 1993). A second problem is that in several instances a high level of basal expression was observed (Furth et al 1994;Howe et al 1995;Kistner et al1996).…”

Section: Tetracyclin-based Inducible Systemsmentioning

confidence: 99%

Changing colors in mice: an inducible system that delivers: Figure 1.

Mills¹

2001

Genes Dev.

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Section: Tetracyclin-based Inducible Systemsmentioning

confidence: 99%

Changing colors in mice: an inducible system that delivers: Figure 1.

Mills¹

2001

Genes Dev.

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“…Currently, the reasons for variable Tet system performance in different cell types are not fully determined, but, in addition to cell type-based variability in efficiency of Ad5 vector transduction, a large factor appears to be the stability of rtTA mRNA and its effect on the levels of the rtTA protein expressed in the cell. 15,29,30 In our study, transduction with the rAd/GFP rTA/TS resulted in induction that varied from 46-fold in A549 to almost 1900-fold in SF295. This performance compares very favorably with previously described transient Tet systems (both plasmid and viral based), which typically achieve between seven-and 350-fold induction.…”

Section: Ad Vectors Deliver Improved Tet Regulation S Rubinchik Et Almentioning

confidence: 56%

New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo

Rubinchik

Woraratanadharm

et al. 2005

Gene Ther

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Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available. Gene Therapy (2005) 12, 504-511.

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“…This inactivity of a single tetO site can also be overcome by multimerization because a reporter construct (7-tetO⅐CMV⅐hGH) containing a heptamer of tetO sites linked to a truncated CMV promoter is activable by tTA and regulated by tetracycline in 266-6 cells and the other cell lines tested (see Table I and Refs. [31][32][33].…”

Section: Resultsmentioning

confidence: 99%

“…Others have shown that tTA transactivation can be cell linespecific (31)(32)(33). We examined the ability of tTA to transactivate in each of the cell lines by cotransfection of the tTA expression plasmid with a reporter gene containing a heptamer repeat of the tetO element linked to the CMV minimal promoter.…”

Section: Resultsmentioning

confidence: 99%

Integration of Tetracycline Regulation into a Cell-specific Transcriptional Enhancer

Rose

MacDonald

1997

Journal of Biological Chemistry

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The pancreas-specific transcriptional enhancer of the rat elastase I gene was modified by substituting, in turn, each of its three individual constitutive elements with the tetO element, which confers regulation by exogenous tetracycline in the presence of the hybrid tetO binding transactivator (tTA). Whereas the unmodified enhancer was active in transfected acinar tumor cells, substitution of individual elements with the tet-responsive element abolished activity. The modified enhancers were reactivated in the presence of the tTA and, upon addition of tetracycline, were silenced. Thus, substitution of individual enhancer elements renders the enhancer responsive to regulation by tetracycline. Moreover, the tTA-activated levels were 2-8-fold greater than the unmodified enhancer. The acinar cell specificity of the unmodified enhancer was retained; none of the tetOsubstituted enhancers were activated by tTA in a variety of nonacinar cell lines. These results show that a foreign and artificial transcriptional activator, tTA, can be incorporated into an enhancer to create a novel, efficient, and regulatable transcriptional control region whose cell specificity is retained.Mammalian transcriptional enhancers and promoters are composed of multiple functional elements of distinct function that contribute to overall transcriptional specificity and strength. In some instances the collection of elements and their bound factors act highly cooperatively; alteration of individual elements or their spacing can dramatically affect the overall activity of an enhancer (1, 2). A precise arrangement of the elements is necessary for the cooperative assembly of DNA binding transcription factors in an ordered nucleoprotein complex required for transcriptional activity. Interactions between bound transcription factors appear crucial as is the presence of DNA-binding proteins whose primary role is to bend DNA to facilitate those interactions (3, 4). The cooperative interaction of transcription factors within this class of enhancer creates a transcriptional activity different than the simple sum of the activities of its individual elements (2, 4). Other enhancers appear to have much more flexible organizational requirements; individual elements play only incremental roles (5, 6) and spacing requirements are much less stringent (7,8). When examined, the separate activities of these enhancers are evident in the individual elements (7-10).One approach to testing the organizational requirements of an enhancer is to examine the activity of each of its elements independently (e.g. Refs. 9 and 11-13). We have used this approach to dissect the functional elements of the transcriptional enhancer of the pancreatic elastase I (EI) 1 gene. The EI enhancer (14) comprises only three mutation-sensitive elements, termed A, B, and C (15, 16) (Fig. 1). By analyzing the elements individually as homomultimers or pairwise combinations of heteromultimers in transgenic mice, we have elucidated the role of each element in controlling the pancreas specificity exh...

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The Responsiveness of a Tetracycline-sensitive Expression System Differs in Different Cell Lines

Cited by 107 publications

References 26 publications

Changing colors in mice: an inducible system that delivers: Figure 1.

Changing colors in mice: an inducible system that delivers: Figure 1.

New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo

Integration of Tetracycline Regulation into a Cell-specific Transcriptional Enhancer

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