1. High throughput QPCR profiling of metabolic, inflammatory, and cancer disease states

Building upon our established HT-QPCR platform we will further extend the NR expression profiling in human and mouse disease models (coordinated with the Mangelsdorf Bridging Project 2 and the Gene Expression Resource).  A range of samples to be profiled include those from human patients with metabolic disorders, cardiovascular disease, environmental toxin exposure and cancer.   We will also explore a variety of related mouse models to examine potential changes in NR expression in response to dietary changes (high-fat, high-cholesterol, high-carbohydrate) associated with obesity, atherosclerosis, hepatic steatosis and insulin resistance.

2. Generation and validation of a murine nuclear receptor (NR) specific-RNAi library

Using on-going protocols for cloning and designing shRNA knockdown sequences we will generate at least 10 different shRNA constructs for each of the 49 mouse nuclear receptors and clone them into a lentiviral shRNA expression vector. The shRNA lentiviral constructs generated will be subjected to a series of cell based knockdown validation experiments to test mRNA expression, protein expression and function in biological assays.  At least 2 individual shRNA knockdown constructs per gene will be obtained that can achieve functional knockdown at a level equal to or greater than 80 percent.  The result will be the generation of a complete validated NR knockdown library in a common lentivirus vector background.

3. Functional targeting of NR pathways in disease

The NR shRNA constructs obtained and validated in Specific Aim 2 will be used to interrogate cell lines and animal models of metabolic, inflammatory and cancer states in which the receptor has been shown to have a dynamic and/or dominant expression profile (Specific Aim 1).  We will also collaborate with other NURSA laboratories (ie. Lazar, Moore and Glass laboratories) to examine the physiological consequences of NR and coregulator knockdown and overexpression.

Information on Ronald Evans' Phase I NURSA Project.