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GENERATION OF TRANSGENIC MICE FOR IN VIVO DETECTION OF INSULIN-CONTAINING GRANULE EXOCYTOSIS AND QUANTIFICATION OF INSULIN SECRETION

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Journal of Innovative Optical Health Sciences 2009年第4期2卷 397-405页

作者： JINLING LU natalia gustavsson QIMING LI GEORGE KRADDA THOMAS C.SUUDHOF WEIPING HAN Laboratory of Metabolic Medicine Singapore Bioimaging Consortium A*STAR Singapore 138667 Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute Stanford University School of Medicine Palo Alto CA 94304USA Department of Biochemistry Yong Loo Lin School of Medicine National University of Singapore Singapore 117597

Insulin secretion is a complex and highly regulated *** much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living *** few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in *** combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in *** this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the *** exocytosis of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory ***,mCherry fluorescence enabled tracking the movement of secretory granules in living *** validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-*** transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.

关键词： Insulin secretion diabetes optical imaging secretory granule exocytosis.

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