Nucleomics: Targeting and Genetic Analysis of Nutrient‐Sensing Cells

Abstract

Pancreatic β‐cells are the most abundant nutrient sensing cell (NSC) type with fewer seen in the hypothalamus and intestine. It is difficult to study NSC's because they are embedded within other cell types in these organs. We showed that glucokinase (GK), a key enzyme in glucose metabolism in β‐cells, is also expressed in the other NSC's. To study NSC's, we developed a gene construct coding for a nuclear targeted fluorescent protein (FP) with a GK promoter to drive expression. Fluorescent nuclei can be rapidly extracted and purified by cell sorting, and the RNA isolated from these nuclei used to evaluate NSC specific gene expression. MIN6 β‐cells were transfected with this plasmid, and targeting of the FP to nuclei of only GK expressing cells was verified by microscopy. Clonal cell lines expressing the construct are now being expanded. Nuclei were isolated after cell disruption, and differential density centrifugation. All nuclei were labeled with a DNA probe DAPI (blue) and all stained nuclei were sorted by FACS, with NSC specific nuclei purified in the FP window. Results show that NSC nuclei and RNA can be isolated away from other cells, thereby providing a method for NSC specific gene analysis. This study will guide development of a mouse line in which changes in β‐cell (and nutrient sensing cells in general) gene expression can be studied during the onset of obesity and diabetes.