A versatile microsatellite instability reporter system in human cells.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Microsatellite instability (MSI) is a hallmark of certain cancers and a sensitive indicator of DNA repair deficiencies.
• Accurate measurement of MSI is crucial for cancer diagnostics and drug screening.

Purpose of the Study:

• To develop and validate a novel, sensitive fluorescent reporter system for quantifying microsatellite instability (MSI) in human cells.
• To investigate the impact of microsatellite characteristics on MSI and identify novel MSI modifiers.

Main Methods:

• Construction of a fluorescence-based reporter vector with variable microsatellites between mCherry and EGFP.
• Introduction of single-copy reporters into HEK293 and MCF-7 cells via targeted recombineering.
• Quantification of EGFP expression using fluorescence-activated cell sorting (FACS) to measure frameshifting events.

Main Results:

• Predominant frameshifts of -1 and +1 base pairs were observed, regulated by mismatch repair.
• Microsatellite tract length and nucleotide composition were found to be major determinants of MSI.
• G-quadruplex formation, strand orientation, and transcriptional status did not significantly affect MSI levels.

Conclusions:

• The developed reporter system is effective for screening compound mutagenicity and identifying MSI modifiers.
• Overexpression of miR-21, targeting MSH2, was identified as a novel inducer of MSI.
• The study demonstrates the utility of combining fluorescent reporters with next-generation sequencing for genetic pathway discovery.