TIF AND ERYTHROLEUKEMIA CELL DIFFERENTIATION

描述：这份修订后的申请描述了研究以澄清 一种新的TIF的功能作用及其分子机制 AXL受体家族的成员。用RT-PCR方法克隆了TIF基因 来自K562文库，是一种非常有趣的受体 它含有细胞外纤维连接蛋白结构域和免疫球蛋白 结构域，暗示在细胞-基质附着和信号传递中发挥作用。 此外，它的RNA表达模式，性腺和脑；其他 组织，暗示了在原始细胞维持中的潜在作用 和自我更新。在本申请书中，申请人建议： 1)对TIF的转化潜力进行了研究 表达；2)研究tif在造血中的作用 用表型分析进行鉴定和鉴别 K562和BA/F3细胞均结构性表达活化的TIF， 通过使用靶向的基因破坏来分化ES细胞 TIF基因座；3)识别紧随其后的下游成分 免疫共沉淀法和酵母菌介导的TIF信号通路 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 5)鉴定和克隆TIF 通过表达或亲和柱层析获得配基。这些 研究旨在为实现以下目标迈出重要的第一步 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.