scholarly journals MipTec Proceedings: Transcriptional Assay Screens Using Renilla Luciferase as an Internal Control

1998 ◽  
Vol 3 (4) ◽  
pp. 41-44
Author(s):  
Rita R. Hannah ◽  
Martha L. Jennens-Clough ◽  
Keith V. Wood
Keyword(s):  
Internal Control ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Imaging System ◽  
Test Sample ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Cell Lysate ◽  
Biology Research ◽  
Assay Conditions

In cell biology research and pharmaceutical discovery, physiological responses of mammalian cells are commonly screened using transcriptional assays. Although firefly luciferase is widely used because of its rapid and simple assay, greater precision can be achieved using a second reporter as an internal control. Renilla luciferase serves as an efficient internal control because it can be measured as easily and rapidly using the same instrument. The Dual-Luciferase™ Reporter (DLR™) Assay developed at Promega measures both reporters sequentially within each sample, which eliminates the need to separate the test sample into aliquots for each assay. The expression of each reporter is independently quantitated using selective assay conditions based on their distinctive chemical characteristics. The firefly luciferase is initiated first by the addition of LAR II to the sample or cell lysate. Following measurement of the luminescent output, the firefly reaction is rapidly quenched and the Renilla reaction is simultaneously activated by addition of Stop & Glo™ Reagent, and the luminescence is measured a second time. The DLR™ Assay allows quantitation of both reporters within 4 seconds per well using a 96-well luminometer equipped with two reagent injectors. Multi-well plates may be processed even more rapidly using a CCD-based imaging system.

scholarly journals Accuracy and precision in quantitative fluorescence microscopy

2009 ◽  
Vol 185 (7) ◽  
pp. 1135-1148 ◽  
Author(s):  
Jennifer C. Waters
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Imaging System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Digital Cameras ◽  
Accuracy And Precision ◽  
Fluorescent Molecules ◽  
Quantitative Fluorescence ◽  
Biology Research

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

Determining the Transrepression Activity of Xenoestrogen on Nuclear Factor-κB in Cos-1 Cells by Estrogen Receptor-α

2007 ◽  
Vol 26 (5) ◽  
pp. 441-449 ◽  
Author(s):  
R. A. Ansari ◽  
J. Gandy
Keyword(s):  
Estrogen Receptor ◽  
Internal Control ◽  
Firefly Luciferase ◽  
Nuclear Factor Κb ◽  
Estrogen Receptor Α ◽  
Binding Activity ◽  
Renilla Luciferase ◽  
Nuclear Factor ◽  
Sv40 Promoter ◽  
Human Estrogen Receptor

Functional assays have been used to define the estrogenicity of xenoestrogens in cotransfection studies employing estrogen receptors in various cell lines. It is known that estrogen is able to affect transcription from other nuclear transcription factors, especially the nuclear factor- κB (NF- κB). The ability of selected xenoestrogens (methoxychlor [MXC], dieldrin, and o′, p′-DDT) to transrepress the NF- κB–mediated transcription in Cos-1 cells was evaluated by cotransfection of human estrogen receptor- α (hER α). These xenoestrogens have been described as comparably potent xenoestrogens, whereas their relative binding activity (RBA) has been relegated to a lower order as compare to estrogen. The two NF- κB response element–containing SV40 promoter and −242/+54 cytomegalovirus (CMV)–expressing firefly luciferase (2 × NRE-PV-Luc and 2 × NRE-CMV-Luc, respectively) were transfected into Cos-1 cells with pRL-tk, expressing the renilla luciferase as internal control. The estrogen receptor was expressed from cytomegalovirus major immediate early promoter (CMV-MIEP) (CMV5-hER α). Treatment with 1 nM estrogen (E2) (26.2%), 5 nM E2 (41.4%; p < .05), and xenoestrogens (methoxychlor [1 nM: 29.6%, p < .05; 10 nM: 22.6%), dieldrin [1 nM: 10.3%; 10 nM: 36.06%, p < .05], and o′, p′-DDT [1 nM: 17.0%; 10 nM: 7.15%]) repressed transcription from 2 × NREX-PV-Luc. The antiestrogen, ICI 182,780, failed to antagonize the effects of xenoestrogens. The effects of xenoestrogens in transrepression of NF- κB by ER α were similar when 2 × NRE-CMV-Luc was employed as reporter. Statistically significant ( p < .01) repression by 1 nM E2 (69.2%), 5 nM E2 (69.1%), 1 nM o′, p′-DDT (51.4%), 1 nM dieldrin (47.3%), and 1 nM MXC (73.3%) were observed. The effect of these xenoestrogens without ER α cotransfection on 2 × NRE-PV-Luc- and 2 × NRE-CMV-Luc-mediated NF- κB transcription was not affected by the treatment alone. It is concluded that xenoestrogens, like estrogens, are capable of producing transrepression of NF- κB by hER α.

Electrokinetic Assembly of Microsphere and Cellular Arrays

2000 ◽  
Vol 662 ◽  
Author(s):  
Mihrimah Ozkan ◽  
Sadik C. Esener ◽  
Sangeeta N. Bhatia
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Electrochemical System ◽  
Charged Species ◽  
Micro Patterning ◽  
Theoretical Predictions ◽  
Oppositely Charged ◽  
Biology Research ◽  
Research Drug ◽  
Cellular Arrays

AbstractWe have developed a novel electrochemical system for field assisted, fluidic assembly of objects on a microfabricated silicon substrate by means of electrical addressing. The principle of our technique is based on the movement of charged species in solution to oppositely charged electrodes, as seen commonly in electrophoresis. Here, charged species such as beads and cells are moved electrokinetically through an aqueous solution towards a charged electrode. Micro patterning of the electrodes allows localization of charged species. We present a theoretical framework to predict the electric potential for assembly and disassembly of spherical objects. We correlate theoretical predictions with the motion of negatively charged polystyrene beads of 20 μm diameter on 100 μm feature micro patterned substrates. In addition, we extended these results to arraying of 20-30 μm diameter live mammalian cells by means of electrical addressing. This technique has applications in creation of ‘active’ cellular arrays for cell biology research, drug discovery and tissue engineering.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 692
Author(s):  
Sweta Talyan ◽  
Samantha Filipów ◽  
Michael Ignarski ◽  
Magdalena Smieszek ◽  
He Chen ◽  
...  
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
De Novo ◽  
Depth Information ◽  
Gene Products ◽  
Classical Analysis ◽  
Protein Coding ◽  
Bioinformatic Pipeline ◽  
Non Coding Rnas ◽  
Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

scholarly journals Biological cell-based screening for scientific membranal and cytoplasmatic markers using dielectric spectroscopy

2008 ◽  
Vol 2 (2) ◽  
pp. 111-121
Author(s):  
Ragini Raj Singh ◽  
◽  
Amit Ron ◽  
Nick Fishelson ◽  
Irena Shur ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dielectric Spectroscopy ◽  
Cell Biology ◽  
Screening Tool ◽  
Excitation Frequency ◽  
Cell Physiology ◽  
Biological Cell ◽  
Non Invasive ◽  
Native Cell ◽  
Biology Research

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of cells suspended in a physiological medium. It provides knowledge of the polarization-relaxation response of the cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of the cells and, therefore, can reveal rare insights into cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. The comparison of the dielectric spectra of tagged and native cell lines reveals clear differences between the cells. In addition, the differences in the matching dielectric properties of the cells were discovered. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.

scholarly journals Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus

2019 ◽  
Author(s):  
Sandy Bauherr ◽  
Filip Larsberg ◽  
Annett Petrich ◽  
Hannah Sabeth Sperber ◽  
Victoria Klose ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Puumala Virus ◽  
Virus Species ◽  
Global Public Health ◽  
Emerging Pathogens ◽  
Cellular Mechanisms

AbstractViruses from the taxonomic familyHantaviridaeare encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with case fatalities of up to 50%. Here we comprehensively investigated entry of the Old-World Hantavirus, Puumala virus (PUUV), into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are significantly reduced. We demonstrated that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, is able to decrease PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV virus particles and showed co-localization of stained virions and markers of macropinocytic uptake. Cells treated with lysosomotrophic agents were shown to exhibit an increased resistance to infection, confirming previous data suggesting that a low pH-dependent step is involved in PUUV infection. Finally, we observed a significant increase in the fluid-phase uptake of cell infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis.Author SummaryTheHantaviridaefamily comprises a very diverse group of virus species and is considered an emerging global public health threat. Human pathogenic hantaviruses are primarily rodent-borne. Zoonosis is common with more than 150,000 annually registered cases and a case fatality index of up to 50%. Individual hantavirus species differ significantly in terms of their pathogenicity, but also their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV virions into mammalian cells. We showed that both, clathrin-mediated endocytosis and macropinocytosis, are cellular pathways exploited by the virus to establish productive infections and demonstrated that pharmacological inhibition of macropinocytosis or its targeted knockdown using RNA interference significantly reduced viral infections. We also found indications for an increase of macropinocytic uptake upon PUUV infections, suggesting that the virus triggers specific cellular mechanisms in order to promote its own internalization and facilitate infections.

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