MOLECULAR ANALYSIS OF THE CYSTIC FIBROSIS LOCUS

囊性纤维化位点的分子分析

基本信息

批准号：
3233185
负责人：
LAP-CHEE TSUI
金额：
$ 10.28万
依托单位：
HOSPITAL FOR SICK CHLDRN (TORONTO)
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-01-01 至 1990-12-31
项目状态：
已结题

项目摘要

The long term objective our research program is to define the basic defect in cystic fibrosis (CF). The approach we have taken initially bypasses the search for a specific defect in the disease. Instead, our aim is to identify DNA markers (probes) closely linked to the disease locus at 7q31 and to then use these markers as reference points to search for the CF gene. Although a number of tightly linked DNA marker have been isolated recently, all available data suggest that there is still a considerable distance between these markers and the CF locus. In this application, we propose to isolate additional DNA markers from the CF region and to use them as probes in combination with pulsed field gel electrophoresis to generate a physical (restriction) map spanning the putative gene locus, from which the CF mutation itself can eventually be identified. Cloned genomic DNA fragments suitable for use as probes will be isolated from the flow sorted chromosome 7-specific library constructed by the Los Alamos and Lawrence Livermore National Laboratories. Each of these probes will be used in hybridization analysis with DNA isolated from a set of human-rodent somatic cell hybrids each containing a subset of human chromosome 7 material spanning the q31 region. A total of 13 DNA probes are now available in our collection. It is estimated that an additional 35-40 DNA probes will be sufficient to saturate the CF region with markers. Restriction fragments length polymorphisms will be identified for each of the probes in the q31 region and studied in a small number of informative families to determine the linkage relationship between these DNA markers and CF. These families have been shown tin our previous studies to contain crossover points near the CF locus. The relative order for those DNA markers that are tightly linked to CF will be further examined by linkage disequilibrium analysis and pulsed field gel electrophoresis. Based on the combined genetic and physical map, it will be possible to initiate chromosome walking from points closet to the CF locus and systematically search for sequences that are expressed in the affected tissues. These experiments will be performed in parallel with other ongoing studies utilizing epithelial cells and tissues that are affected in CF. Specifically, epithelial cell cDNA libraries are being constructed and clones that correspond to genes in the 7q31 region are being identified and tested for their involvement in the disease. Attempts are also being made to establish permanent CF epithelial cell lines in order to develop a functional assay for the CF gene by means of DNA transfection to correct the ion transport defect in these cells. With a combination of these various molecular genetic approaches, the basic defect in CF will be resolved.

我们研究计划的长期目标是确定基本的囊性纤维化（CF）缺陷。我们采取的方法最初绕过了对疾病中特定缺陷的搜索。相反，我们的目标是识别紧密相连的 DNA 标记（探针）到 7q31 的疾病位点，然后使用这些标记作为搜索 CF 基因的参考点。尽管有一些最近分离出紧密连锁的 DNA 标记，所有现有数据表明，仍有相当大的距离这些标记和 CF 基因座之间。在这个应用程序中，我们建议从 CF 区域中分离出额外的 DNA 标记，将它们用作与脉冲场凝胶结合的探针电泳生成物理（限制性）图谱假定的基因位点，CF 突变本身可以从该位点最终被识别。适合用作探针的克隆基因组 DNA 片段将被从流式排序的 7 号染色体特异性文库中分离出来由洛斯阿拉莫斯和劳伦斯利弗莫尔国家体育场建造实验室。这些探针中的每一个都将用于杂交使用从一组人类-啮齿动物体细胞中分离的 DNA 进行分析每个含有人类 7 号染色体材料子集的杂交体跨越 q31 区域。目前共有13个DNA探针在我们的收藏中可用。估计还需要额外加 35-40 个 DNA 探针足以使 CF 区域饱和标记。将鉴定限制性片段长度多态性 q31 区域中的每个探针并进行了少量研究信息家族来确定联系关系这些 DNA 标记和 CF 之间。这些家庭已被展示我们之前的研究可以在 CF 附近包含交叉点轨迹。那些紧密结合的 DNA 标记的相对顺序与CF相关的将通过连锁不平衡进一步检查分析和脉冲场凝胶电泳。基于结合遗传图谱和物理图谱，将有可能启动染色体从最接近 CF 基因座的点行走，系统地搜索在中表达的序列受影响的组织。这些实验将与其他正在进行的实验同时进行利用受影响的上皮细胞和组织进行研究在CF中。具体来说，上皮细胞 cDNA 文库正在构建与 7q31 区域基因相对应的克隆正在对他们参与的情况进行识别和测试疾病。还正在尝试建立永久性CF 上皮细胞系，以开发功能测定 CF基因通过DNA转染的方式来校正离子这些细胞中的运输缺陷。结合这些各种分子遗传学方法，CF的基本缺陷将得到解决。