An extrachromosomal tetracycline-regulatable system for mammalian cells

Sclimenti, Christopher R; Baba, Edward J.; Calos, Michèle P.

doi:10.1093/nar/28.17.e80

Cited by 7 publications

(6 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This has greatly encouraged their use as broad-spectrum antimicrobials, especially in the treatment of chronic infections. They are also currently being used in research to control gene expression in eukaryotes and mammalian cells (41)(42)(43).…”

Section: Structure and Antimicrobial Activity Spectrum Of The Tetracymentioning

confidence: 99%

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

Chukwudi

2016

Antimicrob Agents Chemother

143

View full text Add to dashboard Cite

The tetracycline antibiotics are known to be effective in the treatment of both infectious and noninfectious disease conditions. The 16S rRNA binding mechanism currently held for the antibacterial action of the tetracyclines does not explain their activity against viruses, protozoa that lack mitochondria, and noninfectious conditions. Also, the mechanism by which the tetracyclines selectively inhibit microbial protein synthesis against host eukaryotic protein synthesis despite conservation of ribosome structure and functions is still questionable. Many studies have investigated the binding of the tetracyclines to the 16S rRNA using the small ribosomal subunit of different bacterial species, but there seems to be no agreement between various reports on the exact binding site on the 16S rRNA. The wide range of activity of the tetracyclines against a broad spectrum of bacterial pathogens, viruses, protozoa, and helminths, as well as noninfectious conditions, indicates a more generalized effect on RNA. In the light of recent evidence that the tetracyclines bind to various synthetic double-stranded RNAs (dsRNAs) of random base sequences, suggesting that the double-stranded structures may play a more important role in the binding of the tetracyclines to RNA than the specific base pairs, as earlier speculated, it is imperative to consider possible alternative binding modes or sites that could help explain the mechanisms of action of the tetracyclines against various pathogens and disease conditions.

show abstract

Section: Structure and Antimicrobial Activity Spectrum Of The Tetracymentioning

confidence: 99%

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

Chukwudi

2016

Antimicrob Agents Chemother

143

View full text Add to dashboard Cite

show abstract

“…Regulated expression is being developed so that genes can be turned on or off as needed. [50][51][52] Moreover, recent genetics studies of disc degeneration suggest that genes encoding aggrecan and type IX collagen may be good candidate genes for transfer for modification in strategies to delay or prevent disc degeneration in susceptible populations. 53,54 Recent studies have demonstrated Sox 9 to be an essential transcription factor for type II collagen synthesis as well as chondrogenesis.…”

Section: Future Directionsmentioning

confidence: 99%

Gene therapy for degenerative disc disease

Sobajima

Gilbertson

et al. 2004

Gene Ther

View full text Add to dashboard Cite

Degenerative disc disease (DDD) is a chronic process that can become clinically manifest in multiple disorders such as idiopathic low back pain, disc herniation, radiculopathy, myelopathy, and spinal stenosis. The limited available technology for the treatment of these and other pathologic and disabling conditions arising from DDD is highly invasive (eg, surgical discectomy and fusion), manifesting a certain degree of complications and unsatisfactory clinical outcomes. Although the precise pathophysiology of DDD remains to be clearly delineated, the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, appears to be a final common pathway. It has been hypothesized that imbalance in the synthesis and catabolism of certain critical extracellular matrix components can be mitigated by the transfer of genes to intervertebral disc cells encoding factors that modulate synthesis and catabolism of these components. The successful in vivo transfer of therapeutic genes to target cells within the intervertebral disc in clinically relevant animal models of DDD is one example of the rapid progress that is being made towards the development of gene therapy approaches for the treatment of DDD. This chapter reviews the ability of gene therapy to alter biologic processes in the degenerated intervertebral disc and outlines the work needed to be done before human clinical trials can be contemplated.

show abstract

“…they do not integrate into genomic DNA [5–7]. Hence, it is expected that clone‐specific effects of the genetic neighbourhood (positional effects) on protein expression are avoided [8]. It thus becomes feasible to work with transfected cell pools instead of single cell clones.…”

mentioning

confidence: 99%

“…EBV‐derived single‐plasmid systems for tetracycline‐regulated gene expression have been described previously; these are based on the TetR‐VP16 fusion proteins tetracycline‐controlled transcriptional activator (tTA) [10] or reverse tetracycline‐controlled transcriptional activator (rtTA) [8]. A third system [11] is based on concomitant expression of two fusion proteins, i.e.…”

mentioning

confidence: 99%

“…Thus, relocation of TetR fusion proteins upon binding of inducer can be expected to cause secondary background differences. Pronounced alteration of rtTA expression levels after addition of inducer would promote further differences [8]. Another single‐plasmid EBV system based on regulation by temperature shift (29 °C versus 37 °C) was also expected to display disturbing background differences [14].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Fast set‐up of doxycycline‐inducible protein expression in human cell lines with a single plasmid based on Epstein–Barr virus replication and the simple tetracycline repressor

et al. 2007

View full text Add to dashboard Cite

The function of human proteins is commonly analyzed by heterologous expression in cultured cell lines. Regulated expression, i.e. a system to switch on expression on demand, has clear advantages over constitutive expression. With constitutive expression, cells may die during antibiotic selection because of toxic effects of the expressed protein [1]. Also, for a close match of backgrounds, it is better to compare two states of a single cell line rather than two separately transfected and selected cell lines.Several widely used systems for regulated expression in mammalian cell lines are based on the tetracycline We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on ⁄ off mRNA ratios in the order of 100 : 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on ⁄ off activity ratios were 24 : 1 and 34 : 1, respectively. With enhanced green fluorescent protein, a 23 : 1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on-and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable.Abbreviations CMV, cytomegalovirus; EBV, Epstein-Barr virus; ETTh, ergothioneine transporter from human; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; eGFP, enhanced green fluorescent protein; MPP + , 1-methyl-4-phenylpyridinium; rtTA, reverse tetracycline-controlled transcriptional activator; tetO2, type 2 tetracycline operator; TetR, tetracycline repressor; tTA, tetracycline-controlled transcriptional activator; tTS, tetracycline-controlled transcriptional silencer.

show abstract

An extrachromosomal tetracycline-regulatable system for mammalian cells

Cited by 7 publications

References 12 publications

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

Gene therapy for degenerative disc disease

Fast set‐up of doxycycline‐inducible protein expression in human cell lines with a single plasmid based on Epstein–Barr virus replication and the simple tetracycline repressor

Contact Info

Product

Resources

About