Insulin secretion assays in an engineered MIN6 cell line.

Area of Science:

• * Endocrinology
• * Cell Biology
• * Genetic Engineering

Background:

• * Pancreatic beta cells regulate glucose homeostasis via insulin secretion.
• * The MIN6 cell line is a common model, but its glucose responsiveness declines with passaging.
• * Traditional insulin secretion assays (RIA, ELISA) are costly and time-consuming.

Purpose of the Study:

• * To develop a robust MIN6 subclone (MIN6-6) for high-throughput insulin secretion assays.
• * To enable genome-wide CRISPR screening of insulin secretion.
• * To provide methods for bulk and single-cell insulin secretion analysis.

Main Methods:

• * Development of a MIN6 subclone (MIN6-6) with enhanced glucose responsiveness and Cas9 expression.
• * Establishment of high-throughput nanoLuc assay for bulk insulin secretion quantification.
• * Implementation of single-cell imaging for intracellular insulin granules and exocytosis.
• * Utilization of pooled sgRNA library for genome-wide CRISPR screening.

Main Results:

• * MIN6-6 exhibits high glucose responsiveness and sustained insulin secretion capacity.
• * The nanoLuc assay provides a straightforward method for bulk insulin secretion measurement.
• * Single-cell analysis allows visualization and quantification of insulin granule dynamics.
• * The platform facilitates CRISPR/Cas9 mutagenesis for genetic screens.

Conclusions:

• * MIN6-6 is a versatile beta cell reporter line for advanced insulin secretion studies.
• * The developed methods enable efficient high-throughput and single-cell analysis.
• * This model system supports genetic screens for discovering novel regulators of insulin secretion.