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 fusion protein and the genetically encoded protein calcium sensor, GCaMP6s. GCaMP6s was designed to have high baseline brightness, a wide dynamic range and slow kinetics, making it ideal for the study of calcium flux in -cells (19C21). The use of a genetically encoded fluorescent protein allows the quantification of calcium signaling in live cultures with standard fluorescent microscopy and without the use of dyes. The insulin/luciferase fusion protein is secreted along with insulin as previously reported, allowing simple and inexpensive quantitation of insulin secretion; this approach makes scaling up of high-throughput systems feasible by removing the prohibitive expense of insulin or C-peptide ELISAs (22). The resulting stable cell line is ideal for facilitating the simultaneous quantification of insulin secretion and calcium flux in response to stimuli in both static and perfusion systems. We can then further manipulate this cell line using lentiviral vectors carrying the CRISPRCCas9 system to generate -cells carrying candidate gene mutations. The generation of mutant -cell lines using Casp-8 this system was validated and achieved ~90% efficiency in the deletion of 3 well-described genes critical for -cell function including and and increased expression of the -cell gene luciferase SJ 172550 fusion protein into a single lentiviral vector. These transgenes are driven by the rat insulin promoter (RIP), allowing robust expression in these insulin-producing cells (Fig. 1A). SJ 172550 SJ 172550 The transduction efficiency was >90% (all supplementary material and figures are located in a digital research materials repository (23)). In order to examine the sensitivity of the GCaMP6s calcium reporter, we measured GFP fluorescence intensity in response to different stimuli. Under basal conditions in the absence of glucose, the GFP fluorescence intensity of GCaMP6s is relatively low. This intensity increases 2.5- to 3-fold when cells are incubated in 20 mM glucose. Subsequent exposure to 30 mM KCl to fully depolarize the cells causes green fluorescent protein (GFP) fluorescence intensity to increase ~5-fold over basal levels (Fig. 1B and ?and1C).1C). We validated the use of both of these reporter tools in a perfusion set-up, allowing for an inexpensive and rapid way to measure calcium levels and insulin secretion in the same cultures and at many time points under varying stimuli (Fig. 1C and (23),). Extensive details of this perfusion set-up have been previously published (24). There is good concordance between calcium flux and insulin secretion with a slight kinetic delay in the latter as would be expected considering that calcium signaling is a mediator of insulin secretion. To show that these changes were calcium-dependent, we performed a perfusion in the absence of Ca2+ and with a Ca2+ chelator, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid, and found that both insulin secretion and calcium flux are markedly decreased in response to KCl in the absence of Ca2+ ((23)). The insulinCluciferase fusion protein has been previously shown to be packaged into the endogenous insulin vesicles and secreted from the cell at a 1:1 ratio with insulin and C-peptide upon stimulation with glucose and KCl (25,26). In order to determine if the insulinCluciferase could be used as a simple and inexpensive surrogate for insulin secretion in our EndoC-H1 model system, we performed static glucose-stimulated insulin secretion (GSIS) assays and SJ 172550 assayed the levels of luciferase and C-peptide secreted into the media after each incubation. Luciferase levels strongly correlate with C-peptide levels over a biologically dynamic range of glucose concentrations (Fig. 1D). When comparing basal (2 mM glucose) and stimulated (20 mM glucose), both C-peptide and luciferase levels increase 2.5- to 3.5-fold under stimulated conditions compared with basal levels (Fig. 1E and ?and1F).1F). The luciferase assay remains.
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