scholarly journals Glucose enhances insulin promoter activity in MIN6 β-cells independently of changes in intracellular Ca2+ concentration and insulin secretion

1999 ◽  
Vol 342 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Helen J. KENNEDY ◽  
Imran RAFIQ ◽  
Aristea E. POULI ◽  
Guy A. RUTTER
Keyword(s):  
Promoter Activity ◽  
Photon Counting ◽  
Firefly Luciferase ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Β Cells ◽  
Viral Promoter ◽  
Luciferase Reporter Construct ◽  
Insulin Promoter ◽  
Firefly Luciferase Reporter

Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca2+ concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 β-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca2+ channel inhibitor, verapamil (100 μM). Increases in intracellular [Ca2+] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet β-cells, via a signalling pathway which does not require increases in intracellular [Ca2+] nor insulin release and insulin receptor activation.

scholarly journals Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

2012 ◽  
Vol 18 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Ellen Siebring-van Olst ◽  
Christie Vermeulen ◽  
Renee X. de Menezes ◽  
Michael Howell ◽  
Egbert F. Smit ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Chemical Components ◽  
Simple Liquid ◽  
Luciferase Reporter Construct ◽  
Reagent Cost ◽  
Firefly Luciferase Gene

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

scholarly journals Original article. Transcription factors regulate Forkhead box O1 gene promoter activity in pancreatic β-cells

2011 ◽  
Vol 5 (4) ◽  
pp. 433-439 ◽  
Author(s):  
Ying Luo ◽  
Yan Lin ◽  
Xiao Han
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Screening Method ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Forkhead Box ◽  
Foxo1 Gene

Abstract Background: Transcription factors of the Forkhead box O (Fox O) family have important roles in cellular proliferation, apoptosis, differentiation, and stress resistance. In pancreatic β-cells, FoxO1 protein plays an important role in β-cells development. The molecular mechanism of transcriptional regulation of basal FoxO1 gene expression in pancreatic β-cells is not fully understood. Objectives: Explore the potential transcription factors regulating FoxO1 promoter activity using pancreatic β-cell line (RINm5F cells) Methods: Promoter screening method, luciferase reporter gene analysis, transient expression assay system, and deletion analysis of a -974/-18 bp 5’ upstream region of the mouse FoxO1 gene were used in this study. Results: An inhibition domain (-974/-321) and an activation domain (-321/-18) was identified through deletion analysis of a -974/-18 bp 5’ upstream region of the mouse FoxO1 gene. Using the promoter screening method, several transcription factors were selected. Luciferase reporter studies showed that these factors could regulate FoxO1 promoter activity in RINm5F cells. Among these factors, cAMP response-element binding protein (CREB) could positively regulate FoxO1 promoter activity. Signal transducer and activator of transcription 1 (STAT1) played a negative role on FoxO1 promoter. In addition, ETS oncogene family member Elk-1 did not affect the FoxO1 promoter activity. Conclusion: Two transcription factors (CREB and STAT1) could effectively regulate the mouse FoxO1 gene promoter activity.

scholarly journals The Coding sequence of firefly luciferase reporter gene affects specific hyperexpressionArabidopsis thaliana cpl1mutant

2017 ◽  
Author(s):  
Hisashi Koiwa ◽  
Akihito Fukudome
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Coding Region ◽  
Luciferase Reporter Gene ◽  
Coding Sequence ◽  
Firefly Luciferase Reporter ◽  
Endogenous Genes ◽  
Firefly Luciferase Reporter Gene

AbstractForward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles ofCPL1(Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type andcpl1.Here we show that theLUCcoding sequence is responsible for the high expression incpl1,using a classicalRD29a-LUC. Deletion of theLUC3’-UTR did not change hyperactivation ofLUCincpl1.However, a codon-modifiedLUC(LUC2) produced similar expression levels both in wild type and incpl1. These results indicate that the coding region ofLUCis responsible for thecpl1-specificLUCoverexpression uncoupled with the expression of the endogenous counterpart.

scholarly journals Transient Transfection of the Zoonotic Parasite Babesia microti

Pathogens ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 108 ◽  
Author(s):  
Mingming Liu ◽  
Shengwei Ji ◽  
Mohamed Abdo Rizk ◽  
Paul Franck Adjou Moumouni ◽  
Eloiza May Galon ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Firefly Luciferase ◽  
Transient Transfection ◽  
Babesia Microti ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Transfection Method ◽  
Firefly Luciferase Reporter ◽  
Zoonotic Parasite ◽  
Firefly Luciferase Reporter Gene

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5′-actin promoter, program FA100, and 50 μg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

scholarly journals The distal location of the iron responsive region of the hepcidin promoter

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3436-3437 ◽  
Author(s):  
Jaroslav Truksa ◽  
Pauline Lee ◽  
Hongfan Peng ◽  
Jonathan Flanagan ◽  
Ernest Beutler
Keyword(s):  
Bone Morphogenetic Proteins ◽  
Ex Vivo ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Deficient Diet ◽  
Iron Deficient ◽  
Firefly Luciferase Reporter ◽  
In Vitro Response ◽  
Distal Location

Abstract The response of hepcidin transcription to iron has been repeatedly documented in living mice, but it is difficult to demonstrate the response in ex vivo systems. We have hydrodynamically transfected mice with plasmid constructs composed of a murine hepcidin 1 promoter and fragments of the promoter fused to a firefly luciferase reporter. This method enabled us to quantitate the response of the hepcidin promoter to short-term feeding of a high-iron diet to mice that have been maintained on an iron-deficient diet. We show that the region of the promoter between 1.6 Kb and 1.8 Kb upstream from the start of translation is essential for the response to iron. The promoter region between −260 bp and −1.6 Kb is not essential for the iron responsiveness of hepcidin promoter. The iron-responsive region that we have mapped is the same region required for the in vitro response of HepG2 cells to stimulation with bone morphogenetic proteins and differs from the LPS/IL-6 responsive area.

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