TIF AND ERYTHROLEUKEMIA CELL DIFFERENTIATION

产品说明： 本修订申请描述了阐明 新的抗肿瘤药物tif的功能作用和分子作用机制 Axl受体家族成员。 通过RTPCR方法克隆了Tif基因 从K562库中， 一个非常有趣的受体， 它携带细胞外纤连蛋白结构域以及IG 结构域，表明在细胞基质附着和信号传导中的作用。 此外，其RNA表达模式，性腺>大脑>其他 组织，表明在原始细胞维持中的潜在作用 和自我更新 在本申请中，申请人提出： 1)通过强制过氧化氢法研究tif的转化潜力 2）探讨tif在造血干细胞中的作用 通过分析表型来确定和区分 K562和Ba/F3细胞组成型表达活化的tif， 并通过用靶向基因破坏分化ES细胞， tif基因座; 3）识别直接下游组件 通过免疫单沉淀和通过酵母的TIF信号传导途径 双杂交系统; 4）寻找tif之间的功能相互作用 和Epo受体通过EpoR/tif的强制表达 嵌合体，分析信号转导和细胞表型， 转染Ba/F3和K562细胞;和5）鉴定和克隆tif 通过表达或亲和柱色谱法来纯化配体。 这些 研究旨在提供第一个重要步骤， 了解TIF在造血分化中的作用。

DESCRIPTION: This revised application describes studies to elucidate the functional role and molecular mechanism of action of tif, a new member of the axl family of receptors. Tif, which was cloned by RTPCR from a K562 library, is an extremely interesting receptor in that it bears extracellular fibronectin domains as well as Ig domains, suggesting a role in cell-matrix attachment and signalling. In addition, its pattern of RNA expression, gonad>brain>other tissues, suggests a potential role in primitive cell maintenance and self-renewal. In this application, the Applicant proposes to: 1) Study the transforming potential of tif by enforced over- expression; 2) Investigate the role of tif in hematopoietic determination and differentiation by analyzing the phenotype of both K562 and Ba/F3 cells constitutively expressing activated tif, and by differentiating ES cells with a targeted gene disruption of the tif locus; 3)Identify the immediate downstream components of the tif signaling pathway via immonocoprecipitation and via the yeast two-hybrid system; 4)Search for a functional interaction between tif and the Epo receptor by enforced expression of an EpoR/tif chimera, analyzing signal transduction and cellular phenotype in transfected Ba/F3 and K562 cells; and 5)Identify and clone the tif ligand via expression or affinity column chromatography. These studies are designed to provide the first important steps towards understanding the role of Tif in hematopoietic differentiation.