Application Prospects of Reporter Cell Lines

The reporter cell lines have shown great potential for cell tracing, drug development and gene function studies, etc. Creative Biogene offers reporter cell lines with high sensitivity and accurate localization, which have become a powerful tool of choice for researchers.

What are Reporter Cell Lines and Reporter Genes?

Reporter cell lines are those stably expressed cell lines labeled with reporter genes that can be used to visualize, track and isolate target genes. Reporter cell lines can be generated by exogenous expression driven by the promoter of the target gene with an easily detectable tag (e.g. GFP). However, since the level of exogenous expression may have unpredictable effects on experimental results, researchers prefer to have endogenous expression of reporter genes. Commonly used reporter gene cell lines include GFP Reporter Cell Lines, RFP Reporter Cell Lines, GFP/RFP Reporter Cell Lines, Luciferase Reporter Cell Lines, Dual Reporter Cell Lines, etc.

Applications of Reporter Cell Lines

Researchers use reporter gene assays to study signaling pathways, gene regulation, and the structure of regulatory elements. Stable reporter gene cell lines provide researchers with a highly sensitive and simple method.

Gene expression and promoter research

Cell lines with endogenous marker genes allow researchers to follow protein production and localization in real time in living cells. These cell lines are also suitable for protein blotting, protein isolation and purification, affinity chromatography, immunocytochemistry and flow cytometry. Utilizing these cell lines is also a good solution when there are no good antibodies available. While the endogenous gene is knocked out, the reporter gene is knocked in by homologous recombination to achieve the replacement of the target gene by a reporter gene (e.g. EGFP). After recombination, the promoter of the target gene will regulate the expression of the inserted reporter gene.

For example, to investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK9 cells by integrating the luciferase gene into the genome controlled by the endogenous PGRN promoter. The results showed that luciferase activity was directly correlated with PGRN endogenous promoter activity in the HEK293-PGRN-T2A-Luciferase-KI cell line knock-in cell line.

Protein expression, localization and translocation in cells

Visualization of gene expression can be achieved by adding a tag sequence to the end (or start) of the target gene and generating a fusion protein. We can then use fluorescence to continuously monitor the expression levels of endogenous and recombinant proteins.

Signaling pathway research

To identify effectors of cellular signaling pathways in the context of intact functional cells, cell-based reporter gene assays are ideal. Using pathway-specific promoter response elements downstream of the luciferase reporter gene, signal activity can be conveniently measured by the output of luciferase activity. The system can be applied to a wide range of cell types and signaling pathways and allows high-throughput screening of large libraries of compounds for their inhibitory or activating functions.

Tumor cell detection

Luciferase labeling involves inserting a luciferase gene into the cell genome so that a cell line with luciferase is transferred to a specific mouse to form a model, and injecting luciferin (a substrate for luciferase) into the model mouse causes the luciferase-labeled cells in vivo to glow, thereby observing the specific activity of the tumor cells (without sacrificing the animal).

Drug development research

The use of reporter cell line assays allows for rapid screening of large numbers of samples and the exclusion of compounds that are cytotoxic or do not interact with cells. HEK293 cell lines can be used for drug target identification and candidate evaluation. Once a specific gene is selected as a target, protocols are designed to fuse the target gene with a cellular model of a sensitive reporter gene (e.g., firefly luciferase gene) to produce an easily measurable light signal that reflects changes in target gene transcription. When HEK293 cell lines are treated with appropriate compounds, a light signal reflecting specific changes in gene expression can be generated.

Among them, as an example of using reporter cell lines to assess drug targets, HELA cell lines were labeled with a nuclear protein (Histone 1-mtagBFP2), a cytoskeletal marker (βtubulin-mClover3), and a known autophagy receptor protein (SQSTM1-mRuby3) to screen for kinases that induce the accumulation of cytoplasmic autophagic vesicles inhibitors (with known targets) to assess the accumulation of autophagic vesicles. The researchers treated the cells with two kinase inhibitors, PLK1 inhibitor BI-6727 and PIM kinase inhibitor CX-6258, which increase the number of autophagic vesicles, such as a known autophagy inhibitor, hydroxychloroquine.

Fig. 1 Validation of kinase inhibitors that induce the accumulation of autophagic vesicles in a triple labeled HELA cell line. (Wu, et al., 2018)

Reference:

Wu, Z.; et al. CRISPR/Cas9 Mediated GFP Knock-in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy. Biotechnol J. 2018, Nov;13(11): e1700674.

* For research use only. Not intended for any clinical use.

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Application Prospects of Reporter Cell Lines

What are Reporter Cell Lines and Reporter Genes?

Applications of Reporter Cell Lines

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