ROLE OF FV2 IN ERYTHROPOIESIS

FV2 在红细胞生成中的作用

• 批准号: 6475866
• 负责人: PAUL A NEY
• 金额: $ 22.59万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-15 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6475866
• 关键词: Friend virus; artificial chromosomes; biological signal transduction; cell proliferation; cell transformation; complementary DNA; erythroid stem cell; erythropoiesis; erythropoietin; gene complementation; gene targeting; genetic mapping; genetic strain; genetically modified animals; growth factor receptors; laboratory mouse; nucleic acid sequence; phenotype; receptor expression; transfection /expression vector; viral leukemogenesis; virus genetics; virus protein

Mice infected with Friend virus complex develop acute erythroblastosis, which rapidly progresses to erythroleukemia. The replication defective component of Friend virus complex, SFFV, encodes a mutant retroviral envelope protein (gp55), which confers pathogenicity. Susceptibility to Friend disease is influenced by a number of host factors. One of these, is the Friend virus susceptibility-2 gene (Fv2). Most inbred strains of mice are Fv2 sensitive, except for C57BL6 and related strains which are Fv2 resistant. Fv2 resistance does not prevent retroviral infection or replication. Rather, Fv2 appears to determine whether infected erythroblasts will proliferate in response to gp55. There is evidence that Fv2, gp55, and the erythropoietin receptor (EPOR) are part of a mitogenic complex that is constitutively active and leads to erythroblast proliferation. To test this hypothesis, and to determine the role of Fv2 in erythroid proliferation and transformation, we undertook the positional cloning of Fv2. In our preliminary experiments, we have mapped the Fv2 interval and cloned a contig of 5 bacterial artificial chromosomes (BACs) spanning that interval. We have mapped 10 genes to that interval, including the stem cell kinase receptor (STK). This receptor is a member of the scatter factor family and includes the avian oncogene, v-sea, which causes erythroblastosis in birds. We found that a truncated form of that receptor (SF-STK) is specifically not expressed in Fv2 resistant mice and that expression of SF-STK makes Fv2 resistant mice sensitive to Friend virus, in the setting of T-cell depletion. Therefore, the following experiments are proposed: In specific aim 1, we propose to determine if SF-STK is Fv2. We will do this by producing SF-STK transgenic mice on an Fv2 resistant background, and by knocking out SF-STK expression in Fv2 sensitive mice. In specific aim 2, we take a broad-based approach to identify Fv2. This includes; in vivo complementation with BACs in the Fv2 interval; generation of a transcription map of the Fv2 interval; and evaluation candidate genes molecularly, and in transgenic mice. In specific aim 3, we propose to determine the mechanism of action of Fv2, and the role of Fv2 in erythroid proliferation and transformation. We will accomplish this by knocking out STK (both forms of the transcript) in mice; with biochemical studies of the EPOR complex; and with genetic studies designed to examine the role of signal transduction pathways, downstream of Fv2 and the EPOR, in Friend virus induced proliferation. These studies should improve our understanding of normal hematopoiesis and leukemic transformation.

感染Friend病毒复合体的小鼠可发生急性成红细胞病，并迅速发展为红细胞白血病。Friend病毒复合体的复制缺陷成分SFFV编码一种突变逆转录病毒包膜蛋白（gp55），该蛋白具有致病性。对Friend病的易感性受许多宿主因素的影响。其中之一是Friend病毒易感-2基因（Fv2）。除C57BL6及其相关毒株具有Fv2抗性外，大多数近交系小鼠对Fv2敏感。Fv2耐药性不能阻止逆转录病毒感染或复制。相反，Fv2似乎决定了受感染的红母细胞是否会在gp55的作用下增殖。有证据表明，Fv2、gp55和促红细胞生成素受体（EPOR）是有丝分裂复合体的一部分，该复合体具有组成性活性，并导致红细胞增殖。为了验证这一假设，并确定Fv2在红细胞增殖和转化中的作用，我们进行了Fv2的位置克隆。在我们的初步实验中，我们绘制了Fv2区间，并克隆了跨越该区间的5条细菌人工染色体（BACs）。我们已经将10个基因定位到这个区间，包括干细胞激酶受体（STK）。这种受体是分散因子家族的一员，包括鸟类致癌基因v-sea，它会导致鸟类的红细胞病。我们发现该受体的截断形式（SF-STK）在Fv2抗性小鼠中特异性不表达，并且在t细胞耗竭的情况下，SF-STK的表达使Fv2抗性小鼠对Friend病毒敏感。因此，我们提出以下实验：在具体目标1中，我们提出确定SF-STK是否为Fv2。我们将通过在Fv2抗性背景下生产SF-STK转基因小鼠，以及敲除Fv2敏感小鼠中SF-STK的表达来实现这一目标。在具体目标2中，我们采取广泛的方法来识别Fv2。这包括;在Fv2期与BACs的体内补体；生成Fv2区间转录图谱；并在分子和转基因小鼠中评价候选基因。在具体的目的3中，我们提出确定Fv2的作用机制，以及Fv2在红细胞增殖和转化中的作用。我们将通过敲除小鼠中的STK（两种形式的转录本）来实现这一点；EPOR复合物的生化研究；以及旨在检验Fv2下游信号转导通路和EPOR在Friend病毒诱导增殖中的作用的遗传研究。这些研究应该提高我们对正常造血和白血病转化的认识。