CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme in inflammation-induced prostaglandin E2 (PGE2) production.
• Inhibiting mPGES-1 is a promising therapeutic strategy for treating inflammatory diseases and cancer.
• A lack of efficient high-throughput screening methods hinders the development of mPGES-1 inhibitors.

Purpose of the Study:

• To develop a robust and simple fluorescence imaging strategy for high-throughput screening of mPGES-1 inhibitors.
• To establish mPGES-1 reporter cells using a CRISPR/Cas9 knock-in system.
• To validate the utility of these reporter cells for evaluating mPGES-1 inhibitor efficacy.

Main Methods:

• CRISPR/Cas9 gene editing was employed to create cells with a fluorescent reporter for mPGES-1 expression.
• Reporter cell construction was validated using immunofluorescence, Sanger sequencing, RNA interference (RNAi), and IL-1β stimulation.
• Flow cytometry and fluorescent microplate reader assays were used to quantify fluorescence signal changes in response to mPGES-1 inhibitors.

Main Results:

• Successful generation and validation of mPGES-1 reporter cells were confirmed through multiple experimental approaches.
• Treatment with four known mPGES-1 inhibitors significantly reduced the fluorescence signal intensity in the reporter cells.
• The fluorescence signal changes correlated with inhibitor efficacy, as measured by flow cytometry and microplate reader analysis.

Conclusions:

• The developed fluorescence imaging strategy and mPGES-1 reporter cells offer an efficient and convenient platform for screening and optimizing mPGES-1 inhibitors.
• This approach provides valuable technical support for developing targeted small molecule compounds for anti-inflammatory and anti-cancer therapies.
• The reporter system facilitates the discovery of novel therapeutics targeting the mPGES-1 pathway.