Improved Mammalian Expression Systems by Manipulating Transcriptional Termination Regions

Kim, Dong‐Jun; Kim, Jeong Do; Baek, Kwanghee; Yoon, Yeup; Yoon, Jaeseung

doi:10.1021/bp0341186

Cited by 9 publications

(3 citation statements)

References 17 publications

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“…For example, linking various elements of UAS GAL to a constitutive core results in a functional, galactose inducible promoter [51]. A similar approach has been conducted with regulated regions of the ARO9 UAS [58]. Collectively, these approaches resulted in a library of galactose-inducible promoters with a 40-fold range in induced expression strength, and a tryptophan-inducible promoter with a 29-fold range in induced expression strength.…”

Section: Hybrid Promoter Engineeringmentioning

confidence: 99%

Promoter and Terminator Discovery and Engineering

Deaner

Alper

2016

Synthetic Biology – Metabolic Engineering

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Control of gene expression is crucial to optimize metabolic pathways and synthetic gene networks. Promoters and terminators are stretches of DNA upstream and downstream (respectively) of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. As a result, both of these elements control net protein expression from a synthetic construct. Thus, it is highly important to discover and engineer promoters and terminators with desired characteristics. This chapter highlights various approaches taken to catalogue these important synthetic elements. Specifically, early strategies have focused largely on semi-rational techniques such as saturation mutagenesis to diversify native promoters and terminators. Next, in an effort to reduce the length of the synthetic biology design cycle, efforts in the field have turned towards the rational design of synthetic promoters and terminators. In this vein, we cover recently developed methods such as hybrid engineering, high throughput characterization, and thermodynamic modeling which allow finer control in the rational design of novel promoters and terminators. Emphasis is placed on the methodologies used and this chapter showcases the utility of these methods across multiple host organisms.

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Section: Hybrid Promoter Engineeringmentioning

confidence: 99%

Promoter and Terminator Discovery and Engineering

Deaner

Alper

2016

Synthetic Biology – Metabolic Engineering

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“…The viral CMV promoter and the promoters of mammalian elongation factor 1-alpha (EF1a) and actin genes are routinely used for expression [8][9][10][11]. The following terminators are often used: viral SV40, b-globin, and BGH [12][13][14][15]. In addition, genes are often fused inframe with short epitope tag sequences, such as FLAG, Myc, HA, and V5, at their 5 0 -or 3 0 -terminus to utilize immune detection assays [16,17].…”

Section: Introductionmentioning

confidence: 99%

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

et al. 2015

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Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

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“…The modularity and portability of terminators in mammalian cells has been evaluated in a study that investigated the endogenous human gastrin gene element along with the viral SV40 terminator and affirmed improved reporter gene expression in three tested mammalian cell lines . As a result, this work suggested that mammalian host terminators can be rationally designed in a similar fashion to yeast , for improved translational output.…”

mentioning

confidence: 99%

Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression

et al. 2019

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Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements (i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.

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Improved Mammalian Expression Systems by Manipulating Transcriptional Termination Regions

Cited by 9 publications

References 17 publications

Promoter and Terminator Discovery and Engineering

Promoter and Terminator Discovery and Engineering

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression

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