A Novel Dual-Color Reporter for Identifying Insulin-Producing Beta- Cells and Classifying Heterogeneity of Insulinoma Cell Lines

Abstract

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel “indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS+) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.

Non-coding RNAs derived from an alternatively spliced REST transcript (REST-003) regulate breast cancer invasiveness

“…For qRT-PCR, cDNAs were made from the total RNAs treated with DNase as described in our previous study 42 . Gene-specific q-PCR primer or probe sets ( Table S1 ) for human genes, GAPDH, CyclophilinA, and Actin, and equivalent amounts of cDNA generated as a template were used for qRT-PCR.…”

Investigation of Ultrasound-Mediated Intracellular Ca2+ Oscillations in HIT-T15 Pancreatic β-Cell Line

“…However, when the cells were bathed in HBSS+ containing 11.1 mM glucose (stimulating concentration), the percent of active cells before ultrasound was 10.60 ± 3.21%, and the number was increased by 2-fold with the ultrasound stimulus to 22.72 ± 5.78% (n = 6, p = 0.0025, Figure 1d). Still, the percentages of the active cells were low, which could be due to (1) the heterogeneity of the HIT-T15 cell line [24] and (2) the quantification method, which excluded cells showing Ca 2+ dynamics in low amplitude (less than 2-folds from the basal level).…”

Antiphase Dual-Color Correlation in a Reactant–Product Pair Imparts Ultrasensitivity in Reaction-Linked Double-Photoswitching Fluorescence Imaging

“…AD correlation imaging is completely different from dual-color fluorescence cross-correlation spectroscopy (FCCS). , Dual-color FCCS provides information to calculate equilibrium or rate constants, whereas AD correlation imaging seeks to amplify the sensitivity and reliability of detection. The AD fluorescence has a distinct antiphase and thus is different from fluorescence energy transfer and others ,, as well as the use of two dyes , or fluorescent proteins …”