Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells

Figge, James; Wright, Christopher; Collins, C. B.; Roberts, Thomas M.; Livingston, David M.

doi:10.1016/0092-8674(88)90409-6

Cited by 150 publications

(104 citation statements)

References 24 publications

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“…Hu and Davidson (1987) were the first to use lac elements to control reporter-gene expression reversibly in mammalian cells. Their results were extended by Figge et al (1988), who demonstrated that the lac repressor was able to gain access to the mammalian chromosome to regulate a stably integrated reporter gene.…”

mentioning

confidence: 61%

The lac operator-repressor system is functional in the mouse

Cronin

Gluba

Scrable³

2001

Genes Dev.

144

149

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We report the successful transfer of a fully functional lac operator-repressor gene regulatory system to the mouse. The key component is a lac repressor transgene that resembles a typical mammalian gene both in codon usage and structure and expresses functional levels of repressor protein in the animal. We used the repressor to regulate the expression of a mammalian reporter gene consisting of the tyrosinase promoter embedded with three short lac operator sequences and the tyrosinase coding sequence. Pigmentation of the mouse was controlled by the interaction of the lac repressor with the regulatable Tyrosinase transgene in a manner that was fully reversible by the lactose analog IPTG. Direct control of mammalian promoters by the lac repressor provides tight, reversible regulation, predictable levels of de-repressed expression, and the promise of reversible control of the endogenous genome. The mouse is the most widely used experimental animal to model mammalian development and disease. In recent years, technological advances in genetic manipulation of the mouse genome have made it even more valuable as a research tool, and a great deal of information has been learned from conventional knockout and transgenic experiments. Despite this, it can be difficult to draw definite conclusions from these studies. Embryonic lethality might preclude the possibility of analyzing adult phenotype, or activation of compensatory systems confuse the analysis. Tight, reversible control of gene expression would greatly broaden the possible experimental questions that can be addressed. To gain such control, the ideal system would enable a target gene to be switched on and off repeatedly, without affecting the expression of nontargeted genes. With this ideal in mind, we have developed the lac repressor regulatory system for use in the mouse.The lac operon of Escherichia coli consists of a set of genes coordinately regulated by lactose (Jacob and Monod 1961). The regulatory components of the system are the lac repressor and its DNA-binding sequence, the lac operator. In the absence of lactose, lac repressor occupies the lac operators and prevents transcription. Lactose causes a conformational change in the repressor, and it vacates the operators, allowing RNA polymerase to gain access to the promoter and initiate transcription. Hu and Davidson (1987) were the first to use lac elements to control reporter-gene expression reversibly in mammalian cells. Their results were extended by Figge et al. (1988), who demonstrated that the lac repressor was able to gain access to the mammalian chromosome to regulate a stably integrated reporter gene.Our goal has been to adapt the lac regulatory system to control gene expression in the mouse. In an earlier paper, we reported that transgenes containing the bacterial-coding sequence for the lac repressor downstream of the ␤-actin promoter were heavily methylated and only transcribed in the testis of transgenic mice (Scrable and Stambrook 1997). Methylation and silencing in mice also was observed by W...

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mentioning

confidence: 61%

The lac operator-repressor system is functional in the mouse

Cronin

Gluba

Scrable³

2001

Genes Dev.

144

149

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“…In this system, addition of 5 mM IPTG (isopropyl-␤-Dthiogalactoside) to the culture media allows for regulated expression of the inserted gene product. IPTG is a nonhyrolyzable galactose analog that is non-toxic to NIH 3T3 cells and other mammalian cells at concentrations up to 130 mM (46,47).…”

Section: Caveolin-1 Expression Is Regulated By Oncogenic Stimuli-mentioning

confidence: 99%

Recombinant Expression of Caveolin-1 in Oncogenically Transformed Cells Abrogates Anchorage-independent Growth

Engelman¹,

Wykoff²,

Yasuhara³

et al. 1997

Journal of Biological Chemistry

345

336

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Caveolae are plasma membrane-attached vesicular organelles. Caveolin-1, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells, and muscle cells. Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras (G12V); caveolae are absent from these cell lines. However, its remains unknown whether down-regulation of caveolin-1 protein and caveolae organelles contributes to their transformed phenotype.Here, we have expressed caveolin-1 in oncogenically transformed cells under the control of an inducible-expression system. Regulated induction of caveolin-1 expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that caveolin-1 protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin-1 expression in v-Abl-transformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae as seen by transmission electron microscopy. Consistent with its antagonism of Ras-mediated cell transformation, caveolin-1 expression dramatically inhibited both Ras/MAPK-mediated and basal transcriptional activation of a mitogen-sensitive promoter. Using an established system to detect apoptotic cell death, it appears that the effects of caveolin-1 may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells. In addition, we find that caveolin-1 expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin-1 expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations.

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“…These proteins recognize and bind with very high affinity to specific target sequences, known as operators. It has been shown that prokaryotic repressor-operator systems can also negatively regulate transcription from eukaryotic polII promoters (8,21,22,29).In this study, we evaluated the ability of a prokaryotic repressor-operator system to control the transcription of a D. discoideum tRNA gene in vivo. We used genetic elements which control the expression of tetracycline resistance in bacteria.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

Dingermann¹,

Werner²,

Schütz³

et al. 1992

Mol. Cell. Biol.

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We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO_ operator fragment was inserted at three different positions in front of a tRNAGrU(Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a 1-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 ,g/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in f-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.tRNA suppressors are a classic means of regulating the expression of a protein. In the absence of a suppressor tRNA, the mRNA derived from a gene which has been mutated to contain a stop codon will be incompletely translated. When a tRNA suppressor gene is introduced into the cell, an authentic protein is synthesized, provided the suppressor tRNA inserts the same amino acid as the cognate tRNA of the unmutated codon. This is a powerful tool for analysis of the function of any gene but has been relegated primarily to procaryotes because of the difficulty of manipulating tRNA expression in eukaryotic cells. Eukaryotic tRNA genes contain gene-internal polymerase III (polIII) promoters (for reviews, see references 23 and 46). These transcriptional control regions become part of the mature tRNA coding sequence and are indispensable for tRNA function. As a consequence, these regions are difficult to manipulate. Another consequence is that other than in the case of polII genes, polIII gene promoters cannot be replaced by regulated polII promoters, such as the heat shock and metallothionein promoters.In addition to the gene-internal control elements, 5'-flanking regions of tRNA genes frequently exert a modulatory influence on gene expression (1-3, 10, 15, 17, 27, 37, 41, 47, 49). Although the mechanism of tRNA gene modulation by 5'-flanking regions is not understood, we recently demonstrated that stable binding of a protein near a tRNA gene strongly inhibits its expression (39). This inhibition apparently resulted from masking of the initiation site for tRNA gene transcription by the bound protein and from a strong interaction of the protein with the RNA polIII transcription complex. These findings suggested th...

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Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells

Cited by 150 publications

References 24 publications

The lac operator-repressor system is functional in the mouse

The lac operator-repressor system is functional in the mouse

Recombinant Expression of Caveolin-1 in Oncogenically Transformed Cells Abrogates Anchorage-independent Growth

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

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