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

我们的目标是分离和鉴定巨核细胞， 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 血小板或来自在来自外周血的培养物中生长的巨核细胞 血液或骨髓;（2）逆转录以制备cDNA;（3） 用于富集基因产物的减法程序，所述基因产物仅 在疾病状态下或仅在巨核细胞中表达;（4）克隆 这些特定信息;（5）测序和进一步表征。 每一步都需要进一步的技术发展。我们有 成功地从血小板中分离mRNA，并将此mRNA用作 RT-PCR模板。通过减法进行富集的初步尝试 尚未成功。我们还培养了巨核细胞， 用Ficoll梯度纯化外周血单核细胞， 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。

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.