GENETIC TARGETING OF HEMATOPOIETIC STEM CELLS

许多人类疾病，包括再生障碍性贫血和 骨髓增生综合征是造血干细胞的疾病， （HSC）。 该项目的主要目标是制定一项战略， 在转基因小鼠中靶向HSC的基因表达。这将提供 一种研究正常和慢性炎症重要分子机制的工具， 白血病造血 为了实现这一目标，我们建议 以下具体目标：（1）我们将确定基因组结构 在mSca-1基因座周围的区域中的Ly-6基因簇，和 (2)我们将开发一种系统， 在使用转基因的位点特异性整合的小鼠中， 胚胎干细胞（ES）Sca-1（Ly-6A/E）是一个 Ly-6家族，一个紧密聚集的高度同源基因组 定位于小鼠15号染色体。 我们和其他人注意到， 在转基因小鼠中产生的留下选择标记的突变及其 靶向基因座中的启动子可以导致对细胞的非预期影响。 其他紧密相连的基因的表达。 因此，我们将 表征与Sca-1连接的区域中的鼠Ly-6基因座。 将产生特定的试剂来分析基因的表达 与Sca-1基因座整合位点“相邻”。 将通过分析补充鼠群的表征 人类8号染色体上的一个同线区域，最近发现含有 mLy-6基因的几个潜在同源物。Sca-1“基因敲入”小鼠 正确靶向的ES克隆共表达整合的 用内源Sca-1等位基因转基因。 从本质上讲，长期以来- 小鼠品系中的长期骨髓再生活性 分析驻留在Sca-1+隔室中，该策略应导致 在体内HSC靶向方面。 将使用突变的hCD 4报告基因 在初步原理验证实验中， 以这个载体为目标。 后续实验将包括 将癌蛋白bcl-2和PML/RAR α靶向HSC， 研究该室白血病转化的机制。这 这项工作将在蒂莫西·莱伊博士的监督下进行。 的 实验室在转基因技术和 小鼠造血功能分析。 咨询委员会， 国际公认的实验血液学家， 组装好了 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张病床） 三级保健中心）将提供适当环境， 帮助这位候选人过渡到独立研究。的 这项研究的长期目标是研究 造血和白血病作为一个积极的成员， 血液学/骨髓移植部。

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.