scholarly journals Muscle-specific enhancement of gene expression by incorporation of SV40 enhancer in the expression plasmid

Gene Therapy ◽  
2001 ◽  
Vol 8 (6) ◽  
pp. 494-497 ◽  
Author(s):  
S Li ◽  
F C MacLaughlin ◽  
J G Fewell ◽  
M Gondo ◽  
J Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Plasmid ◽  
Sv40 Enhancer ◽  
Specific Enhancement

scholarly journals CCAAT/enhancer-binding protein β represses human papillomavirus 11 upstream regulatory region expression through a promoter-proximal YY1-binding site

2006 ◽  
Vol 87 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Walter M. Ralph ◽  
Kai Liu ◽  
Karen J. Auborn
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Binding Site ◽  
Binding Protein ◽  
Regulatory Region ◽  
Upstream Regulatory Region ◽  
Expression Plasmid ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Exogenous Expression

CCAAT/enhancer-binding protein β (C/EBPβ) can function as a repressor or as an activator of human papillomavirus (HPV) gene expression, depending on which cell type the experiments are conducted. In this report, it was shown that within primary human foreskin keratinocyte cells (HFK) the activity of C/EBPβ can be switched from that of a repressor of HPV11 expression to an activator by mutating a single promoter-proximal consensus YY1-binding site within the HPV11 upstream regulatory region (URR). It was shown that in HFK cells, exogenous expression of C/EBPβ significantly activates the expression of mutant HPV11 URR reporter plasmids that contain deletions which overlap a 127 bp region (−269 to −142). Inclusive in this region are binding sites for multiple transcription factors, including AP1, YY1 and C/EBPα. Only mutation of the YY1 site resulted in the switch in phenotype, indicating that C/EBPβ represses HPV11 expression in these cells via YY1 binding. The level of YY1 activity was also measured in HFK cells transfected with a C/EBPβ expression plasmid and a significant increase in YY1 activity as compared with mock-transfected cells was found. C33A cells, which exhibit activation of wild-type HPV11 gene expression with exogenous C/EBPβ co-expression, failed to demonstrate C/EBPβ-induced YY1 activation. It was concluded that in HFK cells, exogenous C/EBPβ induces the activity of YY1, which, in turn, can repress HPV11 URR expression through the promoter-proximal YY1-binding site.

scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Author(s):  
Muzi Hu ◽  
Bin Xiong ◽  
Zhongkang Li ◽  
Li Liu ◽  
Siwei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Production ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
Expression Plasmid ◽  
T7 Promoter ◽  
T7 Expression System ◽  
Plasmid Size ◽  
Rna Polymerase Gene ◽  
Electroporation Efficiency

Abstract Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

scholarly journals A novel gene expression system for Ralstonia eutropha based on the T7 promoter

2020 ◽  
Author(s):  
Muzi Hu ◽  
Bin Xiong ◽  
Zhongkang Li ◽  
Li Liu ◽  
Siwei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Production ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
Expression Plasmid ◽  
T7 Promoter ◽  
T7 Expression System ◽  
Plasmid Size ◽  
Rna Polymerase Gene ◽  
Electroporation Efficiency

Abstract Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

Transforming growth factor β-1 enhances Smad transcriptional activity through activation of p8 gene expression

2001 ◽  
Vol 357 (1) ◽  
pp. 249-253 ◽  
Author(s):  
Andrés C. GARCÍA-MONTERO ◽  
Sophie VASSEUR ◽  
Luciana E. GIONO ◽  
Eduardo CANEPA ◽  
Silvia MORENO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Cell Growth ◽  
Mrna Expression ◽  
Transcriptional Activity ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Mouse Embryonic Fibroblasts ◽  
Expression Plasmid ◽  
Embryonic Fibroblasts

We report that exposure of mouse embryonic fibroblasts to transforming growth factor β-1 (TGFβ-1) (5ng/ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8–chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFβ-1 in these cells. The incorporation of [3H]thymidine on treatment with TGFβ-1 was indeed significantly higher in p8+/+ fibroblasts than in p8−/− fibroblasts. Smad transcriptional activity was used as marker of the TGFβ-1 signalling pathway, to probe the lower p8−/− response to TGFβ-1. Two Smad-binding elements (SBEs)–luciferase constructs were transfected into p8−/− and p8+/+ embryonic fibroblasts before treatment with TGFβ-1. A lower level of Smad transactivation was observed in p8−/− embryonic fibroblasts, under basal conditions and after stimulation with TGFβ-1. To test whether Smad underexpression in p8−/− cells was actually due to p8 depletion, p8−/− embryonic fibroblasts were transfected with a human p8 expression plasmid together with an SBE–luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFβ-1-treated cells to the level found in p8+/+ cells. We concluded that TGFβ-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFβ-1 activity.

scholarly journals Development of CRTEIL and CETRIZ, Cre-loxP-Based Systems, Which Allow Change of Expression of Red to Green or Green to Red Fluorescence upon Transfection with a Cre-Expression Vector

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Masato Ohtsuka ◽  
Takayuki Warita ◽  
Takayuki Sakurai ◽  
Satoshi Watanabe ◽  
Hidetoshi Inoko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Cell Lineage ◽  
Green Fluorescence ◽  
Expression Plasmid ◽  
Red Fluorescence ◽  
Gene Switching ◽  
Predominant Expression ◽  
Lineage Analysis

We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre) caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

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