Dataset Information

Quantitative comparison of constitutive promoters in human ES cells.

ABSTRACT:

Background

Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking.

Methodology/principal findings

We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human ?-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1?, (EF1?), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies.

Conclusion/significance

The ACTB, EF1? and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1? promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.

SUBMITTER: Norrman K

PROVIDER: S-EPMC2928720 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Publications

Quantitative comparison of constitutive promoters in human ES cells.

Norrman Karin K Fischer Yvonne Y Bonnamy Blandine B Wolfhagen Sand Fredrik F Ravassard Philippe P Semb Henrik H

PloS one 20100826 8

<h4>Background</h4>Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking.<h4>Methodology/principal findings</h4>We have quantitati ...[more]

PMID: 20865032

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Project description:BackgroundInspiratory patient effort under assisted mechanical ventilation is an important quantity for assessing patient-ventilator interaction and recognizing over and under assistance. An established clinical standard is respiratory muscle pressure [Formula: see text], derived from esophageal pressure ([Formula: see text]), which requires the correct placement and calibration of an esophageal balloon catheter. Surface electromyography (sEMG) of the respiratory muscles represents a promising and straightforward alternative technique, enabling non-invasive monitoring of patient activity.MethodsA prospective observational study was conducted with patients under assisted mechanical ventilation, who were scheduled for elective bronchoscopy. Airway flow and pressure, esophageal/gastric pressures and sEMG of the diaphragm and intercostal muscles were recorded at four levels of pressure support ventilation. Patient efforts were quantified via the [Formula: see text]-time product ([Formula: see text]), the transdiaphragmatic pressure-time product ([Formula: see text]) and the EMG-time products (ETP) of the two sEMG channels. To improve the signal-to-noise ratio, a method for automatically selecting the more informative of the sEMG channels was investigated. Correlation between ETP and [Formula: see text] was assessed by determining a neuromechanical conversion factor [Formula: see text] between the two quantities. Moreover, it was investigated whether this scalar can be reliably determined from airway pressure during occlusion maneuvers, thus allowing to quantify inspiratory effort based solely on sEMG measurements.ResultsIn total, 62 patients with heterogeneous pulmonary diseases were enrolled in the study, 43 of which were included in the data analysis. The ETP of the two sEMG channels was well correlated with [Formula: see text] ([Formula: see text] and [Formula: see text] for diaphragm and intercostal recordings, respectively). The proposed automatic channel selection method improved correlation with [Formula: see text] ([Formula: see text]). The neuromechanical conversion factor obtained by fitting ETP to [Formula: see text] varied widely between patients ([Formula: see text]) and was highly correlated with the scalar determined during occlusions ([Formula: see text], [Formula: see text]). The occlusion-based method for deriving [Formula: see text] from ETP showed a breath-wise deviation to [Formula: see text] of [Formula: see text] across all datasets.ConclusionThese results support the use of surface electromyography as a non-invasive alternative for monitoring breath-by-breath inspiratory effort of patients under assisted mechanical ventilation.

Project description:Streptomycetes attract a lot of attention in metabolic engineering and synthetic biology because of their well-known ability to produce secondary metabolites. However, the available constitutive promoters are rather limited in this genus.In this work, constitutive promoters were selected from a pool of promoters whose downstream genes maintained constant expression profiles in various conditions. A total of 941 qualified genes were selected based on systematic analysis of five sets of time-series transcriptome microarray data of Streptomyces coelicolor M145 cultivated under different conditions. Then, 166 putative constitutive promoters were selected by following a rational selection workflow containing disturbance analysis, function analysis, genetic loci analysis, and transcript abundance analysis. Further, eight promoters with different strengths were chosen and subjected to experimental validation by green fluorescent protein reporter and real-time reverse-transcription quantitative polymerase chain reaction in S. coelicolor, Streptomyces venezuelae and Streptomyces albus. The eight promoters drove the stable expression of downstream genes in different conditions, implying that the 166 promoters that we identified might be constitutive under the genus Streptomyces. Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.This work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus. These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.

Dataset Information

Quantitative comparison of constitutive promoters in human ES cells.

Background

Methodology/principal findings

Conclusion/significance

Publications

Quantitative comparison of constitutive promoters in human ES cells.

Similar Datasets

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