Powerful and versatile enhancer-promoter unit for mammalian expression vectors

doi:10.1016/0378-1119(86)90137-X

Gene

Volume 45, Issue 1, 1986, Pages 101-105

https://doi.org/10.1016/0378-1119(86)90137-X Get rights and content

Abstract

The enhancer and promoter of the immediate early gene of human cytomegalovirus (hCMV) were tested as a transcriptional control element by fusion to thecat gene, followed by measurement of its expression from plasmid DNA in the extrachromosomal and integrated state, respectively. Comparison of the hCMV enhancerpromoter to two other viral elements was performed in six commonly used cell lines of different tissue and species origin. Irrespective of the cell line and of the state of the DNA, the hCMV enhancer-promoter was considerably stronger than both the SV40 promoter and the long terminal repeat of Rous sarcoma virus.

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The mammalian olfactory system consists of sensory neurons with specialized odorant-binding capability accomplished by mutually exclusive odorant receptor (OR) expression. Mutually exclusive OR expression is a complex multi-step process regulated by a number of cis and trans factors, including pan-silencing of all OR genes preceding the robust and stable expression of the one OR selected in each sensory neuron. We transfected two olfactory-placode-derived cell lines modeling immature odorant sensory neurons, as well as the GD25 fibroblast cell line, with episomes containing CMV-driven GFP and TK-driven hygromycin reporter genes. We inserted various coding sequences, along with an IRES, immediately upstream of the GFP gene to produce bicistronic mRNAs driven from the local CMV promoter. We found that the presence of several OR coding sequences resulted in significantly diminished episomal expression of GFP in all three cell lines. These findings suggest that OR coding sequences have intrinsic self-silencing capability that might facilitate mutually exclusive OR expression in olfactory sensory neurons by making it less likely that multiple ORs acquire an above-threshold level of expression at once.
Neuronal Activation Stimulates Cytomegalovirus Promoter-Driven Transgene Expression
2019, Molecular Therapy Methods and Clinical Development
Citation Excerpt :
Due to strong expression and compatibility with numerous cell types, promoters derived from the enhancer region of the cytomegalovirus (CMV) immediate early (IE) promoter are among the most common for general expression vectors.1,2
The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1α (EF1α) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.
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In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.
Improving transcriptional activity of human cytomegalovirus major immediate-early promoter by mutating NF-κB binding sites
2018, Protein Expression and Purification
Citation Excerpt :
When the human and mouse MIEPs were used to produce the monoclonal antibody (mAb) in the Chinese hamster ovary (CHO) cell, the human MIEP had the expression level higher than the mouse MIEP [8]. Originally, the human MIEP has been identified to display high transcriptional activity in a wide range of cultured cells [9]. The human MIEP can drive the ectopic gene expression in broad tissues in transgenic animals [10,11].
Many mammalian gene expression vectors express the transferred genes under the control of the cytomegalovirus (CMV) major immediate-early promoter (MIEP). The human MIEP has been known as the strongest promoter in mammalian cells and utilized widely in mammalian expression systems. There are four NF-κB binding sites (named as κBs) in the human MIEP. In this study, we have constructed multiple mutated MIEPs by changing the natural κBs in the human MIEP into the high-affinity artificial sequences that were in vitro selected by using systematic evolution of ligands by exponential enrichment (SELEX) and predicted by bioinformatics. With various transcriptional activity evaluations, we found three mutated MIEPs with the transcriptional activity higher than the wild-type MIEP, which should be useful and widely applicable in many mammalian transgene expression fields such as gene engineering, gene therapy and gene editing. This study provides a useful approach for promoter engineering in biotechnology. This study also produced a series of mutated MIEPs with various transcriptional activities, which may be used for the fine control of gene expression output in the future synthetic biology.
Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells
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The adenovirus major late promoter (MLP) and its translational regulator – the tripartite leader (TPL) sequence – can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie) promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO) cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds) than translation (11 folds). Even with adenovirus infection to provide additional elements (in trans), CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.
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To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2–1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4–4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.

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Short communicationPowerful and versatile enhancer-promoter unit for mammalian expression vectors

Abstract

Cell

Virology

Cell

Transcriptional “enhancers” from papovaviruses as components of eukaryotic expression vectors

Methods Nucl. Acids Biochem.

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection

Reeombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

Mol. Cell. Biol.

Short communication
Powerful and versatile enhancer-promoter unit for mammalian expression vectors