Functional Cloning of Hutchinson-Gliford progeria gene

Hutchinson-Gliford早衰基因的功能克隆

• 批准号: 6600077
• 负责人: JUNKO OSHIMA
• 金额: $ 15.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6600077
• 关键词: autosomal dominant trait; biomarker; cell line; fibroblasts; flow cytometry; functional /structural genomics; gene complementation; genetic markers; green fluorescent proteins; microarray technology; premature aging; reporter genes; transfection

DESCRIPTION (provided by applicant): The gene for Werner's syndrome, an adult-onset progeroid syndrome, has recently been identified by positional cloning. This approach cannot be used to clone the Hutchinson-Gilford Progeria Syndrome (HGPS) gene because there are no families with HGPS pedigrees. It is therefore proposed to clone the HGPS gene by functional complementation. To clone a gene by functional complementation it is critical to identify a reliable, measurable, phenotypic marker in the diseased cells that is not present in normal cells. But so far, no reliable cellular phenotype has been identified in HGPS. We have recently identified several genes whose expression is markedly upregulated in HGPS cells by expression profiling using high-density oligonucleotide microarrays ("GeneChips"). These genes can now serve as phenotypic markers unique to HGPS fibroblasts. To test the widely held hypothesis that HGPS is the result of an autosomal dominant mutation, HGPS and normal fibroblasts will be fused, and the gene expression profile of the hybrid cells will be analyzed by microarrays. These experiments will also identify those markers that can be used in further studies, namely those genes for which the abnormal level of expression characteristic of HGPS fibroblasts is maintained in the HGPS/normal hybrid lines. To prepare and test cell lines in which the expression of the abnormally expressed genes can be assayed at the single cell level, the promoter regions of the selected marker genes will be fused with reporter genes assayable by fluorescence (such as the green fluorescent protein, or GFP), and stably transfected into normal fibroblasts. Cell lines will be selected in which the expression of the GFP reporter gene(s) mirrors exactly the expression of the endogenous gene(s). To clone the HGPS gene, high titer retroviral cDNA libraries prepared from HGPS cells will be used to infect the cell lines carrying the reporter constructs. Pools of infected cells will be analyzed by flow cytometry and cell sorting to identify cells in which reporter gene expression becomes characteristic of an HGPS/normal hybrid rather than of the normal cell line. Once the putative HGPS gene is identified in this manner, the complete sequence of the gene will be determined in normal and HGPS cells to ascertain the nature of the mutational change.

描述（由申请人提供）：最近通过定位克隆鉴定了沃纳综合征（一种成人型早老性综合征）的基因。这种方法不能用于克隆Hutchinson-Gilford早衰综合征（HGPS）基因，因为没有HGPS家系。因此，建议通过功能互补来克隆HGPS基因。 为了通过功能互补克隆基因，关键是在病变细胞中鉴定出正常细胞中不存在的可靠的、可测量的表型标记。但到目前为止，还没有可靠的细胞表型已确定在HGPS。 我们最近通过使用高密度寡核苷酸微阵列（“GeneChips”）的表达谱分析鉴定了几个基因，其表达在HGPS细胞中显著上调。这些基因现在可以作为HGPS成纤维细胞特有的表型标记。 为了检验HGPS是常染色体显性突变的结果这一广泛持有的假设，将HGPS和正常成纤维细胞融合，并通过微阵列分析杂交细胞的基因表达谱。这些实验还将鉴定可用于进一步研究的那些标志物，即在HGPS/正常杂交系中维持HGPS成纤维细胞特征性的异常表达水平的那些基因。 为了制备和测试其中异常表达基因的表达可以在单细胞水平上测定的细胞系，将所选标记基因的启动子区与可通过荧光测定的报告基因（例如绿色荧光蛋白，或GFP）融合，并稳定转染到正常成纤维细胞中。将选择GFP报告基因的表达与内源基因的表达完全一致的细胞系。 为了克隆HGPS基因，将使用从HGPS细胞制备的高滴度逆转录病毒cDNA文库感染携带报道构建体的细胞系。将通过流式细胞术和细胞分选分析感染细胞的扩增，以鉴定报告基因表达成为HGPS/正常杂交体特征而非正常细胞系特征的细胞。 一旦以这种方式鉴定出推定的HGPS基因，将在正常细胞和HGPS细胞中确定该基因的完整序列，以确定突变变化的性质。