GENETIC TARGETING OF HEMATOPOIETIC STEM CELLS

一些人类疾病，包括再生障碍性贫血和 骨髓增殖性综合征，是一种造血干细胞疾病 (HSC)。这个项目的主要目标是制定一个战略， 在转基因小鼠中靶向HSC的基因表达。这应该会提供 一种研究正常和正常的分子机制的工具 白血病的造血功能。为了实现这一目标，我们建议 具体目标如下：(1)确定基因组组织 在MSCA-1基因座周围区域的Ly-6基因簇，以及 (2)我们将开发一种靶向SCA-1+区基因的系统 在使用定点整合转基因的小鼠中，Via同源基因 胚胎干细胞中的重组。SCA-1(Ly-6A/E)是成员 在Ly-6家族中，一组紧密聚集的高度同源基因 定位于小鼠15号染色体。我们和其他人已经注意到 在转基因小鼠中产生的突变留下了一个可选择的标记和它的 靶基因中的启动子可能导致意外的影响 其他紧密连锁基因的表达。出于这个原因，我们将 鉴定小鼠Sca-1连锁区域中的Ly-6基因座。 将产生特定的试剂来分析基因的表达 “邻近”整合到SCA-1基因座的位置。 对鼠类集群的描述将通过分析加以补充 人类8号染色体上的同线区域，最近发现含有 Mly-6基因的几个潜在同源物。SCA-1“敲入”小鼠来源 来自正确靶向的ES克隆应该共同表达整合的 用内源SCA-1等位基因进行转基因。因为基本上都很长- 不同品系小鼠足月骨髓再生活性的研究 分析驻留在SCA-1+隔间，此策略应会导致 体内的HSC靶向。突变的hc4报告基因将被利用 在初步的原则性实验证明中确立正确 用这个媒介瞄准目标。随后的实验将包括 将癌蛋白bcl2和PML/RARα靶向HSC 研究该隔室白血病转化的机制。这 工作将在蒂莫西·莱伊博士的监督下进行。这个 实验室在转基因技术方面拥有相当多的专业知识， 小鼠造血功能的分析。由以下人员组成的咨询委员会 国际公认的实验血液学家 集合好了。华盛顿大学癌症中心的核心设施 除了华盛顿的科学和临床资源 大学医学院和巴恩斯-犹太医院(1100张床位 三级护理中心)将提供适当的环境 促进候选人向独立研究的过渡。这个 这位研究者的长期目标是研究分子基础 作为临床活跃成员的造血和白血病发生 血液科/骨髓移植科。

A number of human diseases, including aplastic anemia and the myeloproliferative syndromes, are disorders of hematopoietic stem cells (HSC). The primary goal of this project is to develop a strategy for targeting gene expression to HSC in transgenic mice. This should provide a tool to study the molecular mechanisms important for normal and leukemic hematopoiesis. To accomplish this goal, we propose the following specific aims: (1) we will determine the genomic organization of the Ly-6 gene cluster in the region surrounding the mSca-1 locus, and (2) we will develop a system to target genes to the Sca-1+ compartment in mice using site-specific integration of a transgene, a via homologous recombination in embryonic stem (ES) cells. Sca-1 (Ly-6A/E) is a member of the Ly-6 family, a tightly clustered group of highly homologous genes localized to murine chromosome 15. We and others have noted that mutations made in transgenic mice that leave a selectable marker and its promoter in a targeted locus can result in unanticipated effects on the expression of other tightly linked genes. For this reason, we will characterize the murine Ly-6 locus in the region linked to Sca-1. Specific reagents will be generated to analyze the expression of genes "neighboring" the site of integration into the Sca-1 locus. Characterization of the murine cluster will be complemented by analysis of a syntenic region on human chromosome 8, recently found to contain several potential homologs of mLy-6 genes. Sca-1 "knock-in" mice derived from correctly targeted ES clones should coexpress the integrated transgene with the endogenous Sca-1 allele. Since essentially all long- term bone marrow repopulating activity in the strains of mice under analysis resides in the Sca-1+ compartment, this strategy should result in HSC targeting in vivo. A mutated hCD4 reporter gene will be utilized in an initial proof of principle experiment to establish correct targeting with this vector. Subsequent experiments will include targeting the oncoproteins bcl-2 and PML/RAR alpha to HSC in order to study the mechanism of leukemic transformation of this compartment. This work will be conducted under the supervision of Dr. Timothy Ley. The laboratory has considerable expertise in transgenic technology and in the analysis of murine hematopoiesis. An advisory committee consisting of internationally recognized experimental hematologists has been assembled. Core facilities of the Washington University Cancer Center in addition to the scientific and clinical resources of Washington University Medical School and the Barnes-Jewish Hospital (an 1100 bed tertiary care center) will provide an appropriate environment to facilitate this candidate's transition to independent research. The long-term goal of this investigator is to study the molecular bases of hematopoiesis and leukemogenesis as an active member of a clinical Hematology/Bone Marrow Transplant Division.