CORE--HEMATOPOIESIS LABORATORY

曼哈顿综合体提出的几个项目的中心目标 镰刀中心是调节伽马和/或β珠蛋白的合成 对患者的造血细胞进行程序设计以改善患者的 镰状细胞病的症状。温伯格博士的具体目标是 造血核心实验室有：1)提供组织培养方法 以及对组件项目的支持。组织培养方法和技术 在核心实验室中建立和广泛使用的方法包括：a) 甲基纤维素培养用于造血祖细胞的鉴定 (HPC)，b)逆转录病毒介导基因的短期体外培养 干祖细胞的转导和扩增(S/PC)，c)长期 逆转录病毒介导的S/PC细胞转基因培养、扩增及鉴定 S/PC和长期培养起始细胞的测定(LTC-IC)，以及2) 在转导细胞中进行珠蛋白链合成研究 培养、移植转导细胞的小鼠和转基因动物 采用聚丙烯酰胺凝胶电泳法和荧光照相法。 项目、实验和核心的作用如下： 1)项目PI：G.Atweh。实验：HPC的转导和扩增， 分析来自转导细胞的菌落，珠蛋白合成 转导的细胞和克隆。核心的作用：短期液体 转导培养物、两相液体培养物、甲基纤维素培养物 凝胶电泳法和荧光照相法。 2)PI项目：A.银行。实验：HPC的转导和扩增。 长期培养产生的菌落中的转导分析- 启动细胞(LTC-IC)和HPC，细胞合成珠蛋白的分析 和殖民地。核心作用：长期骨髓培养+/-基质，+/- 造血生长因子(HGFs)短期转导培养+/- 基质、凝胶电泳法和荧光照相法。 3)项目PI：F.Constantini：实验：分析珠蛋白链 转基因小鼠克隆中的合成。核心的作用：网织红细胞 孵化、凝胶电泳法、荧光法。 4)项目PI：J.Bieker，实验：用EKLF样锌培养HPC 手指蛋白，分析细胞和克隆中的珠蛋白合成 文化。核心作用：甲基纤维素和两相液体培养，凝胶 电泳法和荧光法 5)PI项目：S.Piomelli。实验：HPC培养+/-精氨酸 丁酸盐，体内前、中和后精氨酸丁酸盐，检查 一群一群地合成珠蛋白。核心角色：甲基纤维素 凝胶电泳法和荧光照相法。

A central goal of several projects proposed by the Manhattan Comprehensive Sickle Center is to modulate the gamma- and/or beta globin synthesis program of hematopoietic cells from patients in order to ameliorate the symptoms of sickle cell disease. The specific aims of Dr. Weinberg's Hematopoiesis Core Laboratory are: 1) To provide tissue culture methods and support to component projects. Tissue culture methods and techniques which are established and used extensively in the core lab include: a) methylcellulose cultures for evaluation of hematopoietic progenitor cells (HPC), b) short term ex vivo cultures for retroviral mediated gene transduction and expansion of stem-progenitor cells (S/PC), c) long term cultures for retroviral mediated gene transduction of S/PC, expansion of S/PC, and assay of long term cultures initiating cells (LTC-IC), and 2) To perform globin chain synthesis studies in transduced cells derived from cultures, mice transplanted with transduced cells, and transgenic animals using polyacrylamide gel electrophoresis and fluorography. Projects, experiments, and core's role are as follows: 1) Project PI: G. Atweh. Experiments: Transduction and expansion of HPC, analysis of colonies derived from transduced cells, globin synthesis by transduced cells and colonies. Role of core: short term liquid transduction cultures, two phase liquid cultures, methylcellulose cultures and gel electrophoresis and fluorography. 2) Project PI: A. Bank. Experiments: Transduction and expansion of HPC. Analysis of transduction in colonies derived from long term culture- initiating cells (LTC-IC) and HPC, analysis of globin synthesis by cells and colonies. Role of core: Long term marrow cultures +/-stroma, +/- hematopoietic growth factors (HGFs), short term transduction cultures +/- stroma, gel electrophoresis and fluorography. 3) Project PI: F. Constantini:Experiment: Analysis of globin chain synthesis in transgenic mice.colonies. Role of core: Reticulocyte incubations, gel electrophoresis, fluorography. 4) Project PI: J.Bieker,Experiments: Culture HPC with EKLF-like zinc finger proteins, analyze globin synthesis in cells and colonies from these cultures. Role of Core: Methylcellulose and two phase liquid cultures, gel electrophoresis and fluorography 5) Project PI: S. Piomelli. Experiments: HPC cultures +/-arginine butyrate, pre-, during, and post- in vivo arginine butyrate, examine globin synthesis by colonies by colonies. Role of core: Methylcellulose and gel electrophoresis and fluorography.