共查询到20条相似文献,搜索用时 31 毫秒
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Knott A Garke K Urlinger S Guthmann J Müller Y Thellmann M Hillen W 《BioTechniques》2002,32(4):796, 798, 800 passim
In addition to the originally described Tet transactivator tTA, several variants including transrepressors (tTRs) and reverse transactivators (rtTAs) have been constructed, which we employ here to establish a set of HeLa cell lines carrying different combinations of chromosomally integrated Tet transregulators. We first compare the regulatory properties of these lines using transient transfection of a luciferase reporter gene. Cell lines carrying rtTA-S2 or rtTA-M2 show reduced activity in the absence of dox and higher activation levels in its presence compared to an rtTA line. rtTA-M2 and its synthetic counterpart rtTA2S-M2 show the same regulation pattern. The replacement of the VP16 activation domain in rtTA-S2 or tTA by p65 leads to slightly reduced expression levels. Combination of an rtTA variant with the transrepressor tTR shows active repression of basal expression without affecting the activation level of the transiently transfected reporter gene. However, if the target gene is also chromosomally integrated, then tTR leads to a further reduction of basal expression and also of the maximal expression level. The results demonstrate that different regulatory windows can be achieved using various transregulators or combinations thereof. Thus, the most appropriate combination of regulators can be chosen depending on the application and cell line desired. We suspect that these properties would also allow the construction of transgenic organisms with preselected expression windows. 相似文献
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Yi Sheng Chih-Cheng Lin Junming Yue Meena Sukhwani Jennifer J Shuttleworth Tianjiao Chu Kyle E Orwig 《BMC developmental biology》2010,10(1):17
Background
The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter. 相似文献4.
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Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator (rtTA-M2). Southern blotting analysis and high-throughput genome sequencing verifed that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline (Dox), a strong green fluorescent protein (GFP) was seen in rtTA-transgenic eggs injected with pTRE--EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox- induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 (DKK1) in pTRE-DKKl-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner. 相似文献
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Xue Zhou Jori Symons Rieuwert Hoppes Christel Krueger Christian Berens Wolfgang Hillen Ben Berkhout Atze T Das 《BMC biotechnology》2007,7(1):6
Background
The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. 相似文献9.
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Qu Z Thottassery JV Van Ginkel S Manuvakhova M Westbrook L Roland-Lazenby C Hays S Kern FG 《Gene》2004,327(1):61-73
Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox. 相似文献
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Mukherjee A Soyal SM Fernandez-Valdivia R DeMayo FJ Lydon JP 《Genesis (New York, N.Y. : 2000)》2007,45(10):639-646
Through an established gene-targeting strategy, reverse tetracycline-dependent transactivator (rtTA) was targeted downstream of the murine progesterone receptor (PR) promoter. Mice were generated in which one (PR(+/rtTA)) or both (PR(rtTA/rtTA)) PR alleles harbor the rtTA insertion. The PR(+/rtTA) and PR(rtTA/rtTA) knockins exhibit phenotypes identical to the normal and the progesterone receptor knockout mouse, respectively. Crossed with the TZA reporter, which carries the TetO-LacZ responder transgene, the PR(+/rtTA)/TZA and PR(rtTA/rtTA)/TZA bigenics exhibit doxycycline-induced beta-galactosidase activity specifically in progesterone responsive target tissues such as the mammary gland, uterus, ovary, and pituitary gland. In the case of the PR(+/rtTA)/TZA mammary epithelium, dual immunofluorescence demonstrated that PR expression and doxycycline-induced beta-galactosidase activity colocalized; beta-galactosidase was not detected in the absence of doxycycline. Although both the PR(+/rtTA) and PR(rtTA/rtTA) knockins represent innovative animal models with which to further query progesterone's mechanism of action in vivo, the PR(rtTA/rtTA) mouse in particular promises to provide unique insight into the paracrine mechanism of action, which underpins progesterone's involvement in mammary morphogenesis with obvious implications for extending our understanding of this steroid's role in breast cancer progression. 相似文献