PROLIFERATIVE SWITCH FOR GENETICALLY MODIFIED CELLS

转基因细胞的增殖转换

• 批准号: 6177913
• 负责人: C. Anthony Blau
• 金额: $ 23.92万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-10 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177913
• 关键词: Retroviridae; animal genetic material tag; bone marrow transplantation; chimeric proteins; developmental genetics; developmental immunology; enzyme activity; erythropoietin; fusion gene; gene induction /repression; genetically modified animals; growth factor receptors; hematopoiesis; hematopoietic stem cells; immunogenetics; immunopharmacology; interleukin 3; laboratory mouse; molecular cloning; oncoproteins; peptidylprolyl isomerase; pharmacogenetics; tissue /cell culture; transfection /expression vector; transplantation immunology

DESCRIPTION: (Adapted from investigator's abstract) The goal of this research is to develop approaches which will allow the proliferative status of genetically modified hematopoietic stem cells to come under pharmacological control. The system involves introducing a gene encoding a fusion protein into hematopoietic cells. The fusion proteins are composed of a cytokine receptor signaling domain linked to one or more copies of the peptide (FKBP12), that permits binding to a drug (FK506) with high affinity. The signaling molecule remains functionally dormant unless forced to engage in dimerization. Dimerization is controlled by adding a dimeric form of FK506, called FK1012, thus mimicking cytokine-activated proliferative signaling. Preliminary studies have used the erythropoietin receptor as a model to show that mitogenic signaling in response to FK1012 can be achieved. Specific Aim 1 will evaluate whether fusion proteins containing the cytoplasmic domains of c-kit or flt-3 can be activated using FK1012. Specific Aim 2 will focus on improving the system by a) minimizing the concentration of FK1012 required to achieve proliferative signaling, b) testing other dimerizers, and c) developing modified receptors in which mitogenic signaling domains are present but maturational signaling domains are absent. Specific Aim 3 will test dimer-inducible proliferative signaling in transgenic mice expressing EpoR/FKBP12 fusion proteins in erythroid cells or c-kit/FKBP12 fusion proteins in stem cells. Stem cells which express c-kit/FKBP12 will be competed against nontransgenic stem cells in the presence of FK1012. Specific Aim 4 will address potential problems in the development of retroviral vectors and test whether FK1012 can promote the selection of retrovirally transduced hematopoietic cells in vitro and in vivo.

描述：（改编自研究者的摘要）本研究的目标 研究的目的是开发允许增殖状态的方法 转基因造血干细胞将受到 药物控制。 该系统涉及引入编码基因 融合蛋白进入造血细胞。 融合蛋白由以下组成 与一个或多个拷贝连接的细胞因子受体信号传导结构域 肽（FKBP12），允许以高亲和力与药物（FK506）结合。 信号分子在功能上保持休眠状态，除非被迫参与 在二聚化中。 通过添加二聚体形式来控制二聚化 FK506，称为 FK1012，因此模仿细胞因子激活的增殖 发信号。 初步研究使用促红细胞生成素受体作为 模型表明响应 FK1012 的促有丝分裂信号传导可以 实现了。 具体目标 1 将评估融合蛋白是否含有 c-kit 或 flt-3 的胞质结构域可以使用 FK1012 激活。 具体目标 2 将侧重于通过以下方式改进系统： a) 最大限度地减少 实现增殖信号传导所需的 FK1012 浓度，b) 测试其他二聚体，以及 c) 开发修饰受体，其中 存在促有丝分裂信号结构域，但存在成熟信号结构域 缺席。 具体目标 3 将测试二聚体诱导增殖 表达 EpoR/FKBP12 融合蛋白的转基因小鼠中的信号传导 红细胞或干细胞中的 c-kit/FKBP12 融合蛋白。 干细胞 表达c-kit/FKBP12的细胞将与非转基因干细胞竞争 在 FK1012 存在的情况下。 具体目标 4 将解决潜在问题 逆转录病毒载体的开发并测试FK1012是否可以促进 体外和体内逆转录病毒转导的造血细胞的选择 体内。