PILOT STUDY--CHARACTERIZATION OF THE MAMMALIAN CAMP-SPECIFIC PHOSPHODIESTERASES

试点研究——哺乳动物营特异性磷酸二酯酶的表征

• 批准号: 6314090
• 负责人: GRAEME B BOLGER
• 金额: $ 7.86万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-15 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6314090
• 关键词: adenosine triphosphate; cell differentiation; cyclic AMP; flow cytometry; gene expression; genetic manipulation; hematopoietic stem cells; leukocytes; neoplastic cell culture for noncancer research; phosphodiesterases; polymerase chain reaction; stimulant /agonist; tissue /cell culture

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.

循环腺苷3'，5'单磷酸（CAMP）的调节很重要 在许多蜂窝过程中，包括学习和记忆以及其他 中枢神经系统中的过程以及造血和 淋巴样细胞及其前体。 我们建议研究功能 哺乳动物细胞中营地特异性磷酸二酯酶（PDES）的 作为 第一个目的，我们将使用同源重组的设施 破坏PC12中编码CAMP特异性PDE的基因的核心 嗜铬细胞瘤细胞系。 已知诱导的代理的影响 PC12细胞的分化（例如神经生长因子，cAMP 类似物或其他）将在这些基因敲除细胞系中进行研究。 这 已知诱导习惯和增强的神经递质的影响 在PC12细胞中（例如ATP或腺苷激动剂）也将进行分析。 我们将研究营地特异性的表达和功能作用 其他细胞系中的PDE，包括源自人血液学的PDE 恶性肿瘤，并在那些显示的线路中尝试类似的淘汰赛 营地特异性PDE表达。 作为第二个目标，我们将净化不同 细胞群，使用流式细胞仪（FACS）和各种单克隆 抗体。 然后，我们将分析营地特异性的表达 这些种群中的PDE，使用逆转录酶/聚合酶 连锁反应。 在要通过茎获得的细胞群中 将使用这些方法分析的细胞核将是各个阶段 在T细胞发育中，由单克隆抗体面板定义。 我们还将分析各种造血细胞前体细胞 种群使用相同的技术。 最后，我们可能会使用差异 从FACS-衍生的mRNA或cDNA上的减法或显示技术 纯化细胞到克隆cDNA的基因，以差异表达 两个不同的细胞群体。