Quantitative real-time analysis of HIV-1 gene expression dynamics in single living primary cells

2006 ◽  
Vol 1 (6) ◽  
pp. 682-689 ◽  
Author(s):  
Asier Sáez-Cirión ◽  
Marie-Anne Nicola ◽  
Gianfranco Pancino ◽  
Spencer L. Shorte
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Primary Cells ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Gene Expression Dynamics ◽  
Single Living ◽  
Hiv 1

Real-time analysis of the transcriptional regulation of HIV and hCMV promoters in single mammalian cells

1995 ◽  
Vol 108 (2) ◽  
pp. 441-455
Author(s):  
M.R. White ◽  
M. Masuko ◽  
L. Amet ◽  
G. Elliott ◽  
M. Braddock ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Sodium Butyrate ◽  
Mammalian Cells ◽  
Viral Gene Expression ◽  
Luciferase Expression ◽  
Viral Gene ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Viral Promoters

The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.

Real-time analysis of GLUT4 trafficking in single living cells using green-fluorescent protein

1997 ◽  
Vol 25 (3) ◽  
pp. 460S-460S
Author(s):  
J.M. Tavaré ◽  
S.P. Dobson ◽  
C. Livingstone ◽  
A.A. Culbert ◽  
P.B. Oatey ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Green Fluorescent ◽  
Single Living Cells ◽  
Single Living

scholarly journals Fluorescence-Based Real-Time Analysis of Osteoclast Development

2021 ◽  
Vol 9 ◽  
Author(s):  
Áron Pánczél ◽  
Simon P. Nagy ◽  
János Farkas ◽  
Zoltán Jakus ◽  
Dávid S. Győri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Real Time ◽  
Bone Marrow Cells ◽  
Green Fluorescence ◽  
Time Analysis ◽  
Green Color ◽  
Real Time Analysis ◽  
Automated Quantification ◽  
Color Conversion

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.

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