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1.
Single-chain Tet transregulators   总被引:3,自引:1,他引:2       下载免费PDF全文
We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.  相似文献   

2.

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   

3.
T R Pacheco  F Maxwell  M F Wu  S Na  I H Maxell 《Gene》1999,229(1-2):125-129
The tetracycline regulatory (TET) system provides a useful means of controlling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100-200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.  相似文献   

4.

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   

5.
To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (P(tets)). We have discovered that stably integrated P(tet) becomes functionally silenced in the majority of neurons when it is inactive during development. P(tet) silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced P(tet) gene silencing, possibly by inducing promoter accessibility.  相似文献   

6.
7.
Stringent doxycycline dependent control of CRE recombinase in vivo   总被引:9,自引:1,他引:8       下载免费PDF全文
The strategy of modulating gene activities in vivo via CRE/loxP recombination would greatly profit from subjecting the recombination event to an independent and stringent temporal control. Here, we describe a transgenic mouse line, LC-1, where the expression of the cre and luciferase gene is tightly controlled by the Tet system. Using the R26R mouse line as indicator for CRE activity, and mouse lines expressing tetracycline controlled transactivators (tTA/rtTA) in various tissues, we show that; (i) in the non-induced state CRE recombinase is tightly controlled throughout the development and adulthood of an animal; (ii) upon induction, efficient recombination occurs in the adult animal in all tissues where tTA/rtTA is present, including hepatocytes, kidney cells, neurons and T lymphocytes; and (iii) no position effect appears to be caused by the LC-1 locus. Moreover, using the novel rTALAP-1 mouse line, we show that in hepatocytes, complete deletion of the loxP-flanked insert in R26R animals is achieved less than 48 h after induction. Thus, the LC-1 mouse appears suitable for exploiting two rapidly increasing collections of mouse lines of which one provides tTA/rtTA in specific cell types/tissues, and the other a variety of loxP-flanked genes.  相似文献   

8.
9.
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   

10.
11.
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   

12.
13.
The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTA CMV (M1) and rTA CMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and -galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTA CMV (M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTA CMV -3/NZL-2 embryos at E13.5. Doxycycline abolished -gal expression in tTA CMV (M1)/NZL-2 but induced it in rTA CMV -3/NZL-2 embryos including late stages of embryogenesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that double reporter animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.  相似文献   

14.
To achieve inducible and reversible gene expression in the adult mouse brain, we exploited an improved version of the tetracycline-controlled transactivator-based system (rtTA2(S)-M2, rtTA2 hereafter) and combined it with the forebrain-specific CaMKIIalpha promoter. Several independent lines of transgenic mice carrying the CaMKIIalpha promoter-rtTA2 gene were generated and examined for anatomical profile, doxycycline (dox)-dependence, time course, and reversibility of gene expression using several lacZ reporter lines. In two independent rtTA2-expressing lines, dox-treatment in the diet induced lacZ reporter expression in neurons of several forebrain structures including cortex, striatum, hippocampus, amygdala, and olfactory bulb. Gene expression was dose-dependent and was fully reversible. Further, a similar pattern of expression was obtained in three independent reporter lines, indicating the consistency of gene expression. Transgene expression could also be activated in the developing brain (P0) by dox-treatment of gestating females. These new rtTA2-expressing mice allowing inducible and reversible gene expression in the adult or developing forebrain represent useful models for future genetic studies of brain functions.  相似文献   

15.
16.
To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.  相似文献   

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20.
Establishing cells with an exogenously introduced gene of interest under the inducible control of tetracycline (Tc) initially requires clonal cell lines stably expressing the tetracycline activator (tTA or rtTA). The originally described plasmid vectors expressing tTA/rtTA are driven by the cytomegalovirus (CMV) immediate early (IE) promoter-enhancer, known for its robust activity in a wide spectrum of cell types. While many reports testify to the utility and efficacy of this construct, instances of inexplicable failure to establish cell lines having inducible expression of the cDNA under study are encountered. Spontaneous extinction of CMV promoter activity in cells has been observed in a temporal and cell type-dependent manner. This could be a contributing factor in the failure to establish Tc-responsive cell lines. We here report that a change of the expression cassette to the human elongation factor-1alpha (EF-1alpha) promoter has permitted successful establishment of several inducible cell lines from diverse human tumor tissue origins. We interpret these results to imply that extinction of rtTA (or tTA) expression might be a significant factor in the lack of success in establishing Tc-inducible cell lines. Moreover, the present findings have general relevance to experiments requiring the use of stable cell lines.  相似文献   

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