This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy
This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPRCCas9 lentiviral system. As a proof of principle, we disrupt the -cell master regulator, PDX1, and show that mutant EndoC-H1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-H1 cells exhibit decreased expression of the -cell-specific genes and and increased expression. The system presented here provides a platform to quickly and easily test -cell functionality in wildtype and cells lacking a gene of interest. luciferase…