ROLE OF FV2 IN ERYTHROPOIESIS

FV2 在红细胞生成中的作用

• 批准号: 6624703
• 负责人: PAUL A NEY
• 金额: $ 23.27万
• 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/6624703
• 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病毒复合物的小鼠发生急性成红细胞增多症，其迅速发展为红白血病。 Friend病毒复合体的复制缺陷组分SFFV编码突变的逆转录病毒包膜蛋白（gp 55），其赋予致病性。 对Friend病的易感性受到许多宿主因素的影响。 Friend virus susceptibility-2 gene（Fv 2）是一种病毒易感基因。 大多数近交系小鼠是Fv 2敏感的，除了C57 BL 6和相关品系是Fv 2抗性的。 fv 2抗性不能阻止逆转录病毒感染或复制。 相反，Fv 2似乎决定感染的成红细胞是否会响应gp 55而增殖。有证据表明，Fv 2，gp 55，和促红细胞生成素受体（EPOR）的一部分，促有丝分裂复合物是组成型活性，并导致成红细胞增殖。 为了验证这一假设，并确定在红系细胞增殖和转化的作用，我们进行了定位克隆的Fv 2。 在我们的初步实验中，我们绘制了Fv 2区间，并克隆了跨越该区间的5个细菌人工染色体（BAC）的重叠群。 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成员，包括导致鸟类成红细胞增多症的禽类致癌基因v-sea。 我们发现该受体的截短形式（SF-STK）在Fv 2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Fv 2抗性小鼠对Friend病毒敏感。 因此，提出以下实验：在具体目标1中，我们提出确定SF-STK是否是Fv 2。 我们将通过在Fv 2抗性背景下产生SF-STK转基因小鼠，并通过敲除Fv 2敏感小鼠中的SF-STK表达来实现这一点。 在具体目标2中，我们采取广泛的方法来识别Fv 2。 这包括：在Fv 2区间中与BAC的体内互补;产生Fv 2区间的转录图谱;以及在转基因小鼠中在分子上评估候选基因。 在具体目标3中，我们提出确定Fv 2的作用机制，以及Fv 2在红系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;对EPOR复合物进行生物化学研究;并进行遗传学研究，旨在研究Fv 2和EPOR下游的信号转导途径在Friend病毒诱导的增殖中的作用。 这些研究应该提高我们对正常造血和白血病转化的理解。