ANALYSIS OF MLL TRANSLOCATIONS AND FUSION GENES

MLL 易位和融合基因分析

• 批准号: 7381572
• 负责人: HEIDI G SUPER
• 金额: $ 16.28万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381572
• 关键词: ANALYSIS; MLL; TRANSLOCATIONS; FUSION; GENES

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Several types of human acute leukemia show chromosome translocations which fuse the MLL gene to numerous other genes. MLL fusion genes are seen in chemotherapy-related leukemia often after treatment with DNA topoisomerase II (topo II)-inhibiting drugs. Two projects of my lab are based on the hypothesis that DNA topoisomerase II (topo II) plays a role in initiating chromosome translocations in acute leukemia involving MLL. Students are examining the translocation breakpoint of the AF4 gene (the most common MLL translocation partner gene) for susceptibility to breakage with topo II inhibitors. In the study, cell lines or cloned bacteria artificial chromosome DNA have been exposed to chemotherapy drugs and DNA has been examined for breakage via topo II. Students have established control conditions for drug activity and DNA analysis. Students are identifying optimal AF4 breakpoint DNA frgments to test, using Vector NTI DNA analysis software and Genbank database sequences. Other students are using chromatin immunoprecipitation (ChIP) to assay the MLL translocation breakpoint region for topo II binding. We have completed pilot experiments to optimize shearing of DNA, the use of positive control antibodies, and PCR analysis of precipitated, end-product chromatin. Students have established conditions for positive control DNA binding proteins. Students are ready to begin the experiments using topo II antibodies, and MLL-specific primers immediately. In a third project, we have designed short interfering RNAs which correspond to the junction of the MLL-AF4 fusion mRNA in the RS4;11 cell line. Our initial aim is to demonstrate specific knock down of expression of the fusion mRNA using RNA interference. Subsequent studies include monitoring the effect on the cell line (growth/transformation changes), longevity of the RNAi effect, and effect on global gene expression. Preliminary experiments have included optimizing transfection (electroporation) conditions for the cell lines.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。几种类型的人类急性白血病表现出染色体易位，将 MLL 基因与许多其他基因融合。 MLL 融合基因常见于化疗相关白血病，通常在使用 DNA 拓扑异构酶 II (topo II) 抑制药物治疗后出现。我实验室的两个项目基于这样的假设：DNA 拓扑异构酶 II (topo II) 在涉及 MLL 的急性白血病中启动染色体易位中发挥作用。 学生们正在检查 AF4 基因（最常见的 MLL 易位伙伴基因）的易位断点对拓扑 II 抑制剂断裂的敏感性。在这项研究中，细胞系或克隆细菌人工染色体 DNA 已暴露于化疗药物中，并通过拓扑 II 检查 DNA 的断裂情况。学生已经建立了药物活性和 DNA 分析的控制条件。学生们正在使用 Vector NTI DNA 分析软件和 Genbank 数据库序列来确定要测试的最佳 AF4 断点 DNA 片段。 其他学生正在使用染色质免疫沉淀 (ChIP) 来检测 MLL 易位断点区域的拓扑 II 结合。我们已经完成了初步实验，以优化 DNA 剪切、阳性对照抗体的使用以及沉淀的最终染色质的 PCR 分析。 学生们已经建立了阳性对照 DNA 结合蛋白的条件。学生们准备好立即使用拓扑 II 抗体和 MLL 特异性引物开始实验。 在第三个项目中，我们设计了短干扰RNA，其对应于RS4;11细胞系中MLL-AF4融合mRNA的连接处。我们最初的目标是证明使用 RNA 干扰对融合 mRNA 的表达进行特异性敲低。随后的研究包括监测对细胞系的影响（生长/转化变化）、RNAi 效应的持续时间以及对整体基因表达的影响。初步实验包括优化细胞系的转染（电穿孔）条件。