scholarly journals Characterisation of a stably integrated expression system for exogenous protein expression in DT40

2017 ◽  
Vol 2 ◽  
pp. 40
Author(s):  
Meliti Skouteri ◽  
Helfrid Hochegger ◽  
Antony M. Carr
Keyword(s):  
Protein Expression ◽  
Transgene Expression ◽  
Expression System ◽  
Biological Processes ◽  
Chromosomal Locus ◽  
Promoter Elements ◽  
Expression Levels ◽  
Exogenous Protein ◽  
Overexpression System ◽  
Dt40 Cells

The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV, CBA or CAG, and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful experimental tool for future DT40 experiments.

scholarly journals Characterisation of a stably integrated expression system for exogenous protein expression in DT40

2017 ◽  
Vol 2 ◽  
pp. 40
Author(s):  
Meliti Skouteri ◽  
Helfrid Hochegger ◽  
Antony M. Carr
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Chromosomal Locus ◽  
Promoter Elements ◽  
Expression Levels ◽  
Beta Actin ◽  
Exogenous Protein ◽  
Cmv Enhancer ◽  
Overexpression System ◽  
Dt40 Cells

The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV (cytomegalovirus), CBA (chicken beta actin) or CAG (a hybrid promoter containing the CMV enhancer and chicken beta actin promoter), and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful tool for future DT40 experiments.

scholarly journals A plasmid-basedEscherichia coligene expression system with cell-to-cell variation below the extrinsic noise limit

2017 ◽  
Author(s):  
Zach Hensel
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Dynamic Range ◽  
Expression System ◽  
Expression Systems ◽  
High Noise ◽  
Gene Expression Noise ◽  
Expression Levels ◽  
Expression Noise ◽  
Negative Autoregulation

AbstractExperiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties inEscherichia colicells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55— 10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range.

High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

1992 ◽  
Vol 68 (02) ◽  
pp. 119-124 ◽  
Author(s):  
F G Falkner ◽  
P L Turecek ◽  
R T A MacGillivray ◽  
W Bodemer ◽  
F Scheiflinger ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Lines ◽  
Expression System ◽  
Human Cell Line ◽  
Cell System ◽  
Time Saving ◽  
Expression Levels ◽  
Mammalian Cell Lines ◽  
Cell Line Hela ◽  
Virus Expression

SummaryWe have worked out an efficient and time saving procedure for the expression of recombinant human prothrombin. The glycoprotein was expressed in the vaccinia virus expression system in several mammalian cell lines. The kidney cell lines Vero and BHK and the human cell line Hela were found to efficiently secrete prothrombin. Expression levels of 3–4 µg of factor II per 106 cells per day corresponding to 18–23 mU per 106 cells per day were achieved. Since the expression levels obtained with the vaccinia virus/Vero cell system were comparable to those obtained in amplified transformed CHO cells it provides an alternative system for the efficient expression of human prothrombin and may allow to further elucidate structure-function relationships of (pro)thrombin and its various effectors.

scholarly journals Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 618
Author(s):  
Yue Jin ◽  
Shihao Li ◽  
Yang Yu ◽  
Chengsong Zhang ◽  
Xiaojun Zhang ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Wild Type ◽  
Expression Levels ◽  
In The Wild ◽  
Cuticle Proteins ◽  
Apolipoprotein D ◽  
High Level ◽  
Lower Expression

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

scholarly journals Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qilu Wei ◽  
Ning Kong ◽  
Xiaohui Liu ◽  
Run Tian ◽  
Ming Jiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fast Green ◽  
Expression Levels ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Safranin O ◽  
Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

scholarly journals Cannabidiol Induces Cell Cycle Arrest and Cell Apoptosis in Human Gastric Cancer SGC-7901 Cells

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 302 ◽  
Author(s):  
Xin Zhang ◽  
Yao Qin ◽  
Zhaohai Pan ◽  
Minjing Li ◽  
Xiaona Liu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Protein Expression ◽  
Cell Cycle Arrest ◽  
Phase Cell ◽  
G1 Phase ◽  
Therapeutic Effects ◽  
Human Gastric Cancer ◽  
Expression Levels ◽  
Cycle Arrest

The main chemical component of cannabis, cannabidiol (CBD), has been shown to have antitumor properties. The present study examined the in vitro effects of CBD on human gastric cancer SGC-7901 cells. We found that CBD significantly inhibited the proliferation and colony formation of SGC-7901 cells. Further investigation showed that CBD significantly upregulated ataxia telangiectasia-mutated gene (ATM) and p53 protein expression and downregulated p21 protein expression in SGC-7901 cells, which subsequently inhibited the levels of CDK2 and cyclin E, thereby resulting in cell cycle arrest at the G0–G1 phase. In addition, CBD significantly increased Bax expression levels, decreased Bcl-2 expression levels and mitochondrial membrane potential, and then upregulated the levels of cleaved caspase-3 and cleaved caspase-9, thereby inducing apoptosis in SGC-7901 cells. Finally, we found that intracellular reactive oxygen species (ROS) increased after CBD treatment. These results indicated that CBD could induce G0–G1 phase cell cycle arrest and apoptosis by increasing ROS production, leading to the inhibition of SGC-7901 cell proliferation, thereby suggesting that CBD may have therapeutic effects on gastric cancer.

scholarly journals Multiple Effects of Genetic Background on Variegated Transgene Expression in Mice

Genetics ◽  
2002 ◽  
Vol 160 (3) ◽  
pp. 1107-1112
Author(s):  
Margaret L Opsahl ◽  
Margaret McClenaghan ◽  
Anthea Springbett ◽  
Sarah Reid ◽  
Richard Lathe ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Milk Protein ◽  
Position Effect ◽  
Position Effect Variegation ◽  
Parental Population ◽  
Expression Levels ◽  
Variable Expression ◽  
Specific Effects ◽  
Milk Protein Genes ◽  
Β Lactoglobulin

Abstract BLG/7 transgenic mice express an ovine β-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA × C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

scholarly journals Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

2021 ◽  
Vol 10 (2) ◽  
pp. 183
Author(s):  
Nadia Meyer ◽  
Lars Brodowski ◽  
Katja Richter ◽  
Constantin S. von Kaisenberg ◽  
Bianca Schröder-Heurich ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Protein Expression ◽  
Tube Formation ◽  
In Vitro Assays ◽  
Heme Oxygenase 1 ◽  
Expression Levels ◽  
Functional Capacities

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction.

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