DETECTION OF MEGAKARYOCYTE-SPECIFIC GENES & GENES ASSOCIATED WITH BONE DISORDERS

巨核细胞特异性基因的检测

• 批准号: 3752283
• 负责人: S ROSENFELD
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3752283
• 关键词: bone disorder; complementary DNA; genetic markers; human tissue; megakaryocytes; messenger RNA; method development; molecular cloning; molecular genetics; nucleic acid sequence; tissue /cell culture

are goal is the isolation and characterization of megakaryocyte- specific gene products in normal and disease states. To this end we plan the following steps: (1) isolation of mRNA from peripheral blood platelets or from megakaryocytes grown in culture from peripheral blood or bone marrow; (2) reverse transcription to make cDNA; (3) a subtraction procedure to enrich for gene products that are only expressed in disease states or only in megakaryocytes; (4) cloning of these specific messages; (5) sequencing and further characterization. Each of these steps involves further technical development. We have successfully isolated mRNA from platelets, and used this mRNA as a template for RT-PCR. Initial attempts to enrich by subtraction have not yet been successful. We have also grown megakaryocytes from peripheral blood mononuclear cells purified over a ficoll gradient, and are currently optimizing methods for confirming the identity of these cells and isolating them at an acceptable purity.

目标是巨核细胞的分离和表征 正常和疾病状态下的特定基因产物。为此我们 计划以下步骤：（1）从外周血中分离mRNA 血小板或来自外周培养物中生长的巨核细胞 血液或骨髓； (2)反转录制备cDNA； （3）一个 扣除程序来富集仅是的基因产物 在疾病状态或仅在巨核细胞中表达； (4) 克隆 这些具体消息； (5)测序和进一步表征。 每个步骤都涉及进一步的技术开发。我们有 成功从血小板中分离出mRNA，并用该mRNA作为 RT-PCR 模板。最初尝试通过减法丰富 尚未成功。我们还培养了巨核细胞 通过聚蔗糖梯度纯化的外周血单核细胞， 目前正在优化确认身份的方法 这些细胞并以可接受的纯度分离它们。