Regulation of cyclic adenosine 3',5' monophosphate (cAMP) is important in many cellular processes, including learning and memory and other processes in the central nervous system, and also in hematopoietic and lymphoid cells and their precursors. We propose to study the function of the cAMP-specific phosphodiesterases (PDEs) in mammalian cells. As a first aim, we will use the facilities of the Homologous Recombination Core to disrupt the genes encoding cAMP-specific PDEs in the PC12 pheochromocytoma cell line. The effects of agents known to induce differentiation of PC12 cells (such as nerve growth factor, cAMP analogues, or others) will be studied in these knockout cell lines. The effect of neurotransmitters known to induce habituation and potentiation in PC12 cells (such as ATP, or adenosine agonists) will also be analysed. We will study the expression and functional role of the cAMP-specific PDEs in other cell lines, including those derived from human hematologic malignancies, and attempts similar knockouts in those lines that show cAMP-specific PDE expression. As a second aim, we will purify different cell populations, using flow cytometry (FACS) with various monoclonal antibodies. We will then analyze the expression of the cAMP-specific PDEs in these populations, using the reverse transcriptase/polymerase chain reaction. Among the cell populations to be obtained by the Stem Cell Core that will be analysed with these methods will be various stages in T-cell development, as defined by panels of monoclonal antibodies. We will also analyze various hematopoietic cell precursor cell populations using the same technology. Finally, we may use differential subtraction or display technologies on mRNA or cDNA derived from FACS- purified cells to clone cDNAs for genes differentially expressed among two different cell populations.

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University of Utah
Salt Lake City
United States
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