Egfp Expression Detection
Egfp Expression Detection - Related Videos
10:03
Development of Leishmania Species Strains with Constitutive Expression of eGFP
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2023
Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.
08:49
Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein
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Cited by 1 •
2011
We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.
10:54
Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression
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Cited by 9 •
2017
In Parkinson's Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.
09:40
Detection of True IgE-expressing Mouse B Lineage Cells
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Cited by 3 •
2014
In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.
03:01
Detection of Target Enzyme Expression in Genetically Modified Bacterial Cells
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2025
Source: Kim, H. et. al., Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System. J. Vis. Exp. (2016)This video demonstrates a genetic screening method to detect enzyme expression using metagenomic fosmid libraries and chromogenic substrates. Flow cytometry identifies and isolates single cells producing colored products, enabling high-throughput selection of active enzyme-expressing cells.
FAQs
Fluorescence does not necessarily appear immediately after EGFP translation because the protein must fold and form its internal chromophore before emitting light. Consequently, early measurements may underestimate activity even when the construct is functioning. Accounting for maturation helps researchers choose meaningful observation times and distinguish delayed reporter development from genuinely weak gene expression.
Fluorescence intensity can vary with EGFP maturation, the physiological state of the cells, and measurement conditions. These variables mean that signal strength is a practical expression readout rather than an isolated measure of construct activity. Comparing samples therefore requires consistent observation conditions and careful interpretation of unusually high or low signals.
Fluorescence microscopy, plate readers, and flow cytometry provide complementary ways to assess EGFP signal. Microscopy allows expression to be observed in cells, whereas plate readers support fluorescence measurements across samples, and flow cytometry can assess signal in cell populations. The appropriate platform depends on whether the study emphasizes visual observation, sample-level measurement, or population-based analysis.
A basic workflow begins with cells carrying the gene or genetic construct, followed by a period that allows EGFP production, folding, and chromophore formation. The cells are then examined using fluorescence microscopy, a plate reader, or flow cytometry. Signal intensity is interpreted alongside maturation, cell state, and measurement conditions to evaluate construct activity.
This readout is useful for characterizing promoters, assessing transfection, studying gene regulation, and tracking engineered cells over time. Each application links green fluorescence to a different experimental question, such as whether a regulatory sequence is active or whether cells received a construct. The same reporter strategy can therefore support both construct evaluation and longitudinal observation.
EGFP fluorescence provides an observable output for testing whether regulatory sequences drive expression under the conditions of an experiment. In promoter characterization, signal helps compare construct activity, while gene regulation studies can use changes in fluorescence as an indicator of altered expression. Interpretation remains dependent on protein maturation, cell state, and measurement conditions.