MAPPING AND MICRODISSECTION OF CHROMSOME 21

该实验室的长期目标是使用细胞和 用分子方法学来研究 人类染色体及其组成基因和调控基因的研究 正常和疾病状态下的序列。 其具体目的是 组成部分的建议是使用体细胞遗传学，分子 探针、显微切割和微克隆技术 人类染色体结构定位、解剖和表达 21. 这些研究将提供直接连接桥接细胞遗传学 和21号染色体的分子分辨率， 寻找病因学的基因和调控序列 唐氏综合症 建议的研究包括（1）绘制 21号染色体使用一系列明确的末端缺失， (2)从染色体的确定区域微克隆DNA序列 21，和（3）显微切割和显微注射的联合使用， 研究培养细胞中大块DNA的表达， 动物 对于末端缺失作图，我们将建立大的 使用人/CHO的21号染色体末端缺失的数量 细胞杂种，含有来自以下的21 q+易位染色体 t（8;21）（q22;q22.3）的AML-M2白血病细胞。 末端缺失 将由X射线产生并由BrdU +可见光选择 使用易位的营养缺陷型标记GlyB的方法 21 q+易位染色体的8 ~ 21号染色体。 大量的DNA探针将用于确定缺失 线性和亚微观分辨率。 这些删除将是 用于所有基因和DNA探针的精确区域定位 分配给21号染色体，并解决许多顺序 紧密相连的基因和DNA标记。 为 显微切割21号染色体，我们将使用新开发的 微切割和微克隆技术。 21号染色体可以是 将分为五个区域，各区域图书馆将 构建用于提供来自所述细胞的限定区域的DNA探针， 用于可能鉴定DNA序列的染色体 患有唐氏综合症 最后，我们将介绍大块的 来自21号染色体切割区域的连续DNA序列 用于研究它们的整合， 表情 这项工作将扩展到转基因小鼠。 这 这种方法对于研究 复杂的疾病比如唐氏综合症

The long-term goal of this laboratory is to use cellular and molecular methodologies to study the organization and expression of human chromosomes and their constituent genes and regulatory sequences in normal and disease states. The specific aim of this component proposal is to use somatic cell genetics, molecular probes, microdissection and microcloning techniques for fine structure mapping, dissection and expression of human chromosome 21. These studies will provide direct link bridging cytogenetic and molecular resolution of chromosome 21 and can facilitate our search for genes and regulatory sequences underlying the etiology of Down syndrome. The proposed studies include (1) mapping of chromosome 21 using a series of well-defined terminal deletions, (2) microcloning of DNA sequences from defined areas of chromosome 21, and (3) combined use of microdissection and microinjection for studying expression of large blocks of DNA in cultured cells and animals. For terminal deletion mapping, we will establish large numbers of terminal deletions of chromosome 21 by using a human/CHO cell hybrid containing a 21q+ translocation chromosome derived from AML-M2 leukemia cells of t(8;21)(q22;q22.3). Terminal deletions will be produced by X-ray and selected by BrdU + visible light method using the auxotrophic marker GlyB which was translocated from chromosome 8 to 21 of the 21q+ translocation chromosome. Large numbers of DNA probes will be used to define the deletions in linear and submicroscopic resolution. These deletions will be used for refined regional mapping of all genes and DNA probes assigned to chromosome 21, and for resolving the order of many closely linked genes and DNA markers on this chromosome. For microdissecting chromosome 21, we will use the newly developed microdissection and microcloning techniques. Chromosome 21 can be subdivided into five regions and region-specific libraries will be constructed for providing DNA probes from defined areas of the chromosome for possible identification of DNA sequences associated with Down syndrome. Finally we will introduce large blocks of contiguous DNA sequences from dissected regions of chromosome 21 into cells by microinjection for studying their integration and expression. This work will be extended to transgenic mice. This approach appears particularly important for studying expression of complex disorders like Down syndrome.