Understanding human health problems requires a detailed functional understanding of the various organs and tissues within the body. These organs and tissues comprise mixtures of different cell types, and obtaining a functional understanding of these cell types at the molecular level is impeded by their complex pattern of interspersion. In this project, our main biomedical goal is to derive information about the global transcriptional and epigenetic states within the different cells of the pancreas, and to particularly identify changes as these cells enter into neoplasia resulting in pancreatic ductal adenocarcinoma (PDAC). The main technical goal of this project is to solve the problem of identifying the neoplastic cells, and characterizing their transcriptional and epigenomic states, within a background of normal cells of various types. A general solution to this problem is proposed based on the following observations: (a) that global profiling of nuclear RNA transcripts provides an accurate description of RNA transcript levels within the cell, (b) that nuclei can be labeled fluorescently using the Green Fluorescent Protein (GFP) within specific cell types of transgenic organisms, (c) that Green Fluorescent nuclei can be purified from cell-free organ and tissue homogenates, using flow cytometry and fluorescence-activated sorting, and (d) that the nuclei can also provide an appropriate source of information concerning epigenomic modifications that impact gene expression. This solution will be specifically evaluated by focusing on oncogenesis induced in the pancreas in established mouse models of PDAC. To achieve transgenic GFP labeling of the nuclei of cells entering PDAC neoplasia, transgenic mouse lines will be produced that respond to the Cre recombinase within pancreatic precursor cells by producing nuclear- targeted GFP. These will be mated to existing mouse lines that express the Cre recombinase within specific cell types, and further to mice that also respond to the Cre recombinase with the production of the KrasG12D oncogene. Progeny mice will be employed for the production of cell-free pancreatic homogenates, and Green- Fluorescent nuclei will be purified by fluorescence-activated sorting. The transcripts in the sorted nuclei will be employed for global transcript profiling using Next Generation sequencing, and methods will be developed and employed to chart the epigenomic status of the chromatin in these nuclei. New experimental protocols will be developed and new information derived for cell type-specific global expression profiles which will be provided freely to the research community. The relevance of this research to public health is two-fold: (1) to provide a uniquely detailed understanding of how human organs function at a cellular level and (2) to provide a fuller understanding of cellular modifications that may have profound implications for disease states.
The relevance of this research to public health is that, through the development and application of novel biological technologies, it will provide a uniquely detailed understanding of how human organs function, based on discovering the biological and molecular functions of all the different cells that make up these organs. This will lead to a fuller understanding of diseases that affect organs, and should accelerate progress in the search for drugs that alleviate and cure human disease.