We have recently discovered that inorganic-organic nanocomposites prepared from TiO2 and DNA oligonucleotides have the unique new property of site-specific light-/radiation-induced-nucleic acid endonucleases. This means that these nanocomposites can be used for precise dissection of only a single gene in the whole genome in a time-dependent manner. These experiments are designed to determine parameters that are needed in order to harness unique properties of these nanocomposites. As a model gene target for nanocomposite cleavage we will use Proliferating Cell Nuclear Antigen (PCNA), a housekeeping gene that is necessary for cell survival and that is overexpressed in a large number of tumors. This gene has been studied in our laboratory using a wide variety of different approaches. In these experiments we propose: 1) To use nanocomposites to cleave the promoter of the PCNA gene driving expression of a reporter gene in cells in culture. We will use a double stably transfected cell line with two reporter genes (ECFP and EYFP) driven by two versions of the PCNA promoter, and we will try to abolish expression from only one of these promoters. 2) To use nanocomposites to cleave mRNAs for two reporter proteins in a sequence specific/selective manner. 3) To develop thymus-specific nanocomposites and to use them to abolish PCNA gene expression in a selected mouse tissue in order to cause thymus ablation.
This aim will test the ability of the nanoparticles to be targeted to a particular tissue in a whole animal as well as their ability to function in that tissue. Our hypothesis is that TiO2-DNA nanocomposites can be used to turn off gene expression both in vivo and in situ. Furthermore, the experiments we propose here will uncover the role of PCNA gene expression in the maintenance of thymic cell population in normal and wasted mice. Accomplishment of the aims of this proposal will most importantly establish the use of nanocomposites for gene therapy and provide a starting point for developing tools for """"""""nanotherapy"""""""". In addition, the experiments as designed will help us learn about the functional significance of PCNA expression in cells and tissues and possible applications of nanoparticles to whole animal imaging.
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