The reporter cell line is a stable cell line that has been labeled with a reporter gene, which can realize the visualization and tracking of protein expression. The reporter gene is inserted next to the promoter of the gene of interest in the vector and transfected into the cell line. If the promoter is expressed in the cell line, the reporter gene will also be expressed, so that cell metabolism, transcription factors and protein localization can be monitored. Common reporter genes include luciferase and fluorescent proteins, such as GFP and RFP. When choosing a product, one should consider the origin of the cell line, the expected application and the reporter gene.
Reporter genes include luciferase and fluorescent proteins, such as GFP and RFP. Creative Biogene has developed a receptor cell line containing different antibiotic selection markers, puromycin, neomycin or elastin. It also provides validated luciferase reporter gene cell lines selected from a variety of host cell lines. These cell lines can measure the activity of transcription factors as a readout of various signal pathways.
The new reporter cell line can be used in many fields, such as in vitro monitoring of HIV infection and drug susceptibility. In vitro determination of HIV infectivity and suppression by antiretroviral drugs by monitoring the reduction in p24 antigen production are expensive and time-consuming. Such a measurement also does not allow accurate quantification of the number of infected cells over time. In order to develop a simple, rapid and direct method to monitor HIV infection, a stable T cell line (CEM) was generated, which contains a plasmid encoding green fluorescent protein (humanized S65T GFP) driven by HIV-1 long terminal repeats.
The selected clones showed low constitutive background fluorescence, but showed high levels of GFP expression after HIV infection. Monitoring by fluorescence microscopy, cell fluorescence and flow cytometry, HIV-1 infection causes a 100 to 1,000-fold increase in relative cell fluorescence within 2 to 4 days. Adding reverse transcriptase inhibitors, proteases, and other targets for different infection multiplicity can accurately determine drug sensitivity.
In vitro monitoring of HIV infection and antiretroviral drug activity is usually performed by detecting infected cells or HeLa-CD4 plaque assay to detect the production of p24 HIV core antigen. However, these methods require multiple operations, are time consuming, and are expensive for the p24 antigen. In addition, the p24 assay can measure the total production of viral proteins in cultured cells and cannot accurately quantify the number of infected cells. Cytotoxicity assays that measure reduction of tetrazolium have similar limitations.
To further study the role of viral transcription in GFP expression, the researchers used UV-psoralen light-inactivated HIV-1 strain to challenge the cells. Under these experimental conditions, no increase in fluorescence expression was detected, confirming the necessity of virus replication for GFP expression. In addition, the expression of HIV-1 gp120 protein on the cell surface is also related to the increase of GFP expression.
Potential applications of the CEM-GFP reporting system include high-throughput assays for anti-HIV drug susceptibility testing or neutralization assays. The method is fast (1 to 6 days depending on the amount of inoculation), simple (direct detection of the fluorescence of HIV-infected cells by cytofluorescence), and inexpensive (essentially maintenance of the cells). The 96-well plate format allows the analysis of multiple drugs within a certain concentration range. The feasibility of performing reverse transcriptase, gag, tat and protease inhibitor drug assays has been demonstrated to be comparable to the results obtained by p24 assay and HeLa-CD4 plaque assay. In addition, two isolates were analyzed, one was sensitive to zidovudine from the same person before and after treatment, and the other was highly resistant to zidovudine.
In conclusion, the establishment of a stable cell line expressing the LTR-driven GFP reporter gene provides a fast, simple, cheap and accurate tool for monitoring and studying HIV infection, anti-HIV drug activity and T cell signaling. Compared with traditional methods, visualization, direct counting, and the unique features of sorting fluorescent cells without adding external substrates or labels are also valuable.