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How do reporter-labeled cell lines work?
Reporter cell lines are common stable cell lines that have been labeled with different kinds of reporter genes. In the field of research, reporter-labeled cell lines are very useful tools for analyzing gene expression and for screening cell lines.
The technique of attaching fluorescent dyes to the functional groups that are present in biomolecules is known as fluorescent labeling. This allows the biomolecules to be observed via the use of fluorescence imaging. The introduction of newly discovered fluorophores has brought about a revolutionary shift in the scope of options for the sensitive detection of biomolecules and the investigation of the interactions between them.
The development of better fluorescent dyes has made it feasible to study cellular processes and structures, something that was previously impossible. Fluorescent labels have numerous benefits to offer, including the fact that they are very sensitive even at low concentrations, that they are stable over extended periods of time, and that they do not interfere with the function of the molecules that they are labeling.
Targeted imaging of cell lines that have been tagged makes it possible to follow the cell lines both in vitro and in vivo. When several fluorophores are used in the same sample, it is possible to see many molecules at the same time. This can be done if you use different fluorophores. The fluorophores fluorescein isothiocyanate (FITC), derivatives of rhodamine (TRITC), coumarin, and cyanine are the ones that are used the majority of the time. Labeling biomolecules such as proteins, peptides, antibodies, nucleic acids, yeast, or bacteria may be accomplished with the use of these synthetic organic dyes.
It is also possible to genetically identify live cell lines by using fluorochromes that exist naturally in the environment, such as the Green Fluorescent Protein (GFP).
Fluorescent protein labeling
Researchers are able to explore the conformational dynamics and molecular interactions of proteins by the use of fluorescent labeling, as well as monitor the motions of proteins, in order to get a better understanding of their biological activities of proteins.
Labeling individual peptides is another method that may be used to investigate protein structures, enzyme activities, and the interactions between receptors and their ligands. Fluorochrome-labeled antibodies are an important component of immunodiagnostics and are used extensively in the field of biomedical research for the purpose of detecting antigens in immunofluorescence experiments. Fluorophore conjugation should not interfere with the features of the antibody that are responsible for its ability to bind to an antigen if trustworthy findings are to be obtained.
Fluorescent labeling of nucleic acids
Assays that rely on fluorescence are very important components of biophysical investigations into the structure, function, and dynamics of nucleic acids.
Recent advancements in labeling technologies and imaging equipment have made it possible to directly see DNA and RNA in vivo, as well as their interactions with many other cell components. This was previously impossible. The imaging of nucleic acids in live cell lines has opened up new avenues for improving our knowledge of chromatin architecture and the control of gene expression.
Fluorescent labeling of polysaccharides
Components of the extracellular matrix that are responsible for its structural integrity include complex polysaccharides like heparin. These polysaccharides are required for cellular adhesion, migration, and proliferation in order to be successful (Prigent-Richard S. et al, 1998).
The anticoagulant, antithrombotic, anti-inflammatory, antiviral, and antiangiogenic effects of several substances are well documented. Methods that rely on fluorescence make it simpler to recognize newly discovered bioactive polysaccharides and to define the biological roles of these molecules.
Fluorescent labeling of lipids
Cellular lipids are very important for the cell since they are responsible for the storage of energy, the development of cellular membranes, and the process of intracellular signaling.
Nile red, a lipophilic dye, is often used in order to stain intracellular lipids for the purpose of determining their localization and structure (Greenspan P et al, 1985). In addition, for the purpose of researching the dynamics of lipids, specific labeling in live cell lines is also a possibility.
How can this system be utilized?
This system focuses on measuring whether particular promoter expression exists, but it can be used to assess the activities of different biological systems which have downstream signals ending with the transcription of genes.
The ability to identify which gene is present can also determine whether cells or cell lines have been infected and suggest the existence of cancers. It's therefore very helpful when studying various diseases as it gives easily interpretable results as to how genes can be expressed under various conditions.
More advantages of fluorescence-tagged cell lines are:
In vivo imaging
Methods for fluorescent labeling
To dock with their targets, fluorophores undergo chemical modifications (covalent or non-covalent binding). There are several benefits to using chemical labeling techniques since they are reliable, simple to use, and very effective with many different types of fluorophores. As a result, in vitro experiments are preferable to in vivo ones.
Techniques for enzymatic labeling
Rapid, highly effective, and selective labeling of proteins or entire cell lines can be achieved using enzymatic processes in both in vivo and in vitro settings. However, the labels' huge sizes might cause interferences with the performance of the target molecules.
Tagging of peptides/proteins
Proteins may now be labeled in a targeted manner without compromising the structure or function of the molecule thanks to a newly discovered, very promising technology that employs short fluorescent tags.
Depending on the specificity of the peptide tag, this straightforward method may be utilized to probe unique protein locations.
Fluorescently labeled protein domains, peptides, or amino acids may be used for genetic tagging since they can bind to particular places along chromosomes in vivo. Using this method, chromosomal anomalies like deletions and duplications may be identified.
The capacity to track or detect numerous fluorescently tagged proteins simultaneously is a standard need for live cell line imaging and flow cytometry applications.
Dyes having a significant Stokes shift are useful for this because they enable several biological reactions to be monitored at once.
Assays based on fluorescence detection
The capacity of fluorophores to re-emit light upon exposure to light particles or photons is fundamental to fluorescence-based tests. In optical microscopes and imaging systems, the Stokes shift, the difference in wavelength between excitation light and emission light, may be measured.
The Stokes shift of each individual fluorophore is unique. Several tests enable scientists to pinpoint the precise location of biomolecules, monitor them in real-time, probe their relationships, and analyze their enzymatic activity.
With the use of fluorescence microscopy, cell lines and their components may be identified and cellular physiology can be closely monitored.
Optical filters are used in fluorescence microscopy to separate fluorescent emission from fluorescent excitation. Since two indicators are used, many biomolecules may be observed in real-time. However, modern super-resolution fluorescence microscopes like STED (stimulated emission depletion) bypass these constraints and reveal insights into the nanoscale realm of molecules, which were previously inaccessible with traditional imaging techniques owing to resolutions of just 200 to 300 nm (Sanderson MJ et al, 2014).
Flow cytometry measures the signal from individual fluorophores and has many applications in both academic and clinical settings. Cells and particles are "real-time" examined and sorted as they go through the laser beam of detectors that measure the fluorescence emitted by tagged antibodies or ligands. These markers may be used for the detection and quantification of target molecules on the cell surface or inside the cell by binding to them.
The size and volume of individual cells may be determined, and various cell types can be extracted and analyzed (Nolan JT and Condello D, 2013). Flow cytometry has many different uses in many areas of study, including but not limited to immunology, hematology, transplantation medicine, cancer, and genetics.
Different microarray technologies have made it possible to examine gene expression in a wide range of settings. DNA chips allow for the simultaneous analysis of thousands of genes.
The DNA sequences printed on these microscopic slides are designed to attach specifically to fluorescently tagged mRNA or cDNA. The DNA chip is read after hybridization, and the information is utilized to compile gene expression profiles (Hoen PAC et al, 2003).
Fluorescence in situ hybridization (FISH)
Through a technique called fluorescence in situ hybridization (FISH), genetic markers may be pinpointed to exact locations on chromosomes. DNA or RNA probes that fluoresce in the presence of their corresponding target DNA sequences are utilized for this purpose. During the Human Genome Project and before, FISH was employed to locate genes on chromosomes. Current applications of fluorescent in situ hybridization include the study of cancer cell lines and the identification of chromosomal aberrations (O'Connor C, 2008).
Fluorescence correlation spectroscopy (FSC)
By using fluorescence correlation spectroscopy (FCS), researchers may examine fluctuations in fluorochromes' fluorescence across time that result from chemical, biological, or physical processes. FCS was first developed to examine drug-DNA interactions and has now evolved into a sensitive instrument for measuring protein concentration and aggregation and for monitoring molecular interactions (Tian Y et al, 2011).
There are several factors that can influence the likelihood of successful identification of endogenous proteins by a fluorescent label. Labeling endogenous proteins can be toxic for proteins that cause misfolding and dysfunction.
In case the protein in question has a low expressed value it can be possible to create fluorescent labels successfully but cannot detect fluorescence from the low expression of the protein of interest.
Most common reporter-labeled cell lines
CRISPR-NUP96-mEGFP clone no.195
This cell line has been endogenously tagged in both alleles of the C-terminus with mEGFP using CRISPR/Cas9D10A NUP96.
CRISPR-NUP96-Halo clone no.252
This cell line has been endogenously tagged in both alleles of the C-terminus with a Halo-Tag using CRISPR/Cas9D10A NUP96. It has been transfected with UniProtKB; P0A3G2; Rhodococcus rhodochrous dhaA (mutated = Halo-Tag).
U-2 OS-CRISPR-NUP96-mMaple clone no.16
This cell line has been endogenously tagged in both alleles of the C-terminus with mMaple using CRISPR/Cas9D10A NUP96. It has been transfected with UniProtKB; Q9U6Y3; GFP-like fluorescent chromoprotein cFP484 (with many modifications leading to the photoconvertible protein mMaple).