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: 6239221
• 负责人: GRAEME B BOLGER
• 金额: $ 10.11万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-05 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6239221
• 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.

环腺苷 3',5' 单磷酸 (cAMP) 的调节很重要 在许多细胞过程中，包括学习和记忆等 中枢神经系统以及造血系统的过程 淋巴细胞及其前体细胞。 我们建议研究一下这个函数 哺乳动物细胞中 cAMP 特异性磷酸二酯酶 (PDE) 的研究。 作为 第一个目标，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。 已知诱导剂的作用 PC12细胞分化（如神经生长因子、cAMP 类似物或其他）将在这些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作用 PC12 细胞中的（例如 ATP 或腺苷激动剂）也将被分析。 我们将研究 cAMP 特异性的表达和功能作用 其他细胞系中的 PDE，包括源自人类血液学的细胞系 恶性肿瘤，并尝试在那些显示的细胞系中进行类似的敲除 cAMP 特异性 PDE 表达。 作为第二个目标，我们将纯化不同的 细胞群，使用流式细胞术（FACS）和各种单克隆抗体 抗体。 然后我们将分析 cAMP 特异性的表达 使用逆转录酶/聚合酶计算这些人群中的 PDE 连锁反应。 Stem获得的细胞群中 将使用这些方法分析的 Cell Core 将处于不同阶段 T 细胞发育中，如单克隆抗体组所定义。 我们还将分析各种造血细胞前体细胞 使用相同技术的人群。 最后，我们可以使用微分 对源自 FACS 的 mRNA 或 cDNA 进行消减或展示技术 纯化细胞以克隆 cDNA，以获取差异表达基因 两个不同的细胞群。