High-Throughput DNA Microarrays for Osteoblasts
DESCRIPTION (provided by applicant): This Phase II grant proposes to screen for 100,000 compounds that stimulate differentiation of adult human mesenchymal stem cells using a multiple mRNA assay, in 96-well plates. The assay quantifies induction of genes that signal differentiation to osteoblasts, chondrocytes, and other connective tissue types. The same approach was validated in Phase I, analyzing the time course and dose-response for gene induction upon differentiation of murine multi-potential cells to osteoblasts and myoblasts. Further, the approach worked well in a screen of 1760 chemical compounds. This Phase II grant proposes the use of a quantitative high-throughput gene chip assay, ArrayPlate TM, which applies nuclease protection protocols to gene chips. Briefly, cells are grown in 96 well plates with test compounds in each well, and are lysed in the presence of oligonucleotides that hybridize to and protect targeted mRNA sequences. After nuclease digestion, samples are transferred to microarray plates, and oligonucleotides that had bound mRNA targets stoichiometricaUy are quantified. Sixteen genes are tested per sample. The assay is sensitive, specific and reproducible, with uncertainties of 3-13 % (CVs). As few as 150,000 mRNA molecules can be measured, and rare and highly expressed genes can be accurately quantified in one sample. This Phase II grant will apply a novel assay that screens both for activity and selectivity by quantifying a pattem of gene expression to a newly identified cell type, adult human mesenchymal stem cells. These have much therapeutic potential for osteoporosis and osteoarthdtis. We propose one efficient method for exploiting these cells to find compounds that induce differentiation of osteoblasts (for treating osteoporosis) or chondrocytes and connective tissue cells (for treating degenerative diseases such as osteoarthritis).
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NEOGEN, LLC BOX 64326 TUCSON, AZ 85728
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