Pilot & Feasibility Program Application Abstract
Nucleomic Analysis of Cell-type Specific Gene Expression in Diabetic Mouse Models
Ronald Lynch (Tucson, AZ)
Many of the metabolic changes that underlie the development and complications of Diabetes are related to dysfunction of pancreatic beta-cells. Beta-cells are the most abundant nutrient sensing cell (NSC) type with fewer NSC’s seen in the hypothalamus and intestine. It is difficult to study these cell types because they are embedded within other cell types in these organs. Moreover, a range of mouse lines have been developed to mimic or parallel the pathophysiological processes observed in response to Diabetes. Therefore, development of methods to rapidly evaluate changes in gene expression and function of the effected cells will greatly facilitate the usefulness of these models systems. We, and others, showed that glucokinase (GK), a key enzyme in glucose metabolism in beta-cells, is also expressed in the other NSC’s. We previously showed that a fluorescent protein (FP) could be specifically targeted to NSC’s in mice expressing a GK promoter driven GFP construct. However, expression of GFP was low, and turnover high making it difficult to use these mice for NSC analysis. To this end, we developed a gene construct coding for a nuclear targeted FP with a histone binding domain and the GK promoter to drive expression. The mouse insulinoma cell line MIN6 was transfected with this plasmid, and targeting of the FP to nuclei of only GK expressing cells was verified by microscopy. Moreover, fluorescent levels remained high in the nuclei even after isolation. On a first order basis, we propose that GK driven-FP constructs can be used to identify nutrient sensing cells within complex tissues for detailed pheno-typing studies. Importantly however, fluorescent nuclei can be rapidly extracted from tissue and purified by cell sorting, and the RNA isolated from these nuclei used to evaluate NSC specific gene expression. We have shown that NSC nuclei and RNA can be isolated away from nuclei of other cell types, thereby providing a method for NSC specific gene analysis. We propose to develop mouse lines in which promoters for specific cell types relevant to the development of diabetes and its complications are used to drive expression of nuclear targeted FP’s. Mating of these mice with others exhibiting outcomes of Diabetes will provide a general methodology for analysis of gene expression in pancreatic beta-cells and other NSC’s during the development and treatment of this disease.
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