Functional Cloning of Hutchinson-Gliford progeria gene

Hutchinson-Gliford早衰基因的功能克隆

• 批准号: 6747941
• 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/6747941
• 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）：最近通过位置克隆确定了Werner综合征的基因，一种成人发作的后代综合征。这种方法不能用来克隆Hutchinson-Gilford Forperia综合征（HGP）基因，因为没有HGPS血统的家庭。因此，提议通过功能互补来克隆HGP基因。 要通过功能互补来克隆基因，至关重要的是，在正常细胞中不存在的患病细胞中识别可靠的，可测量的表型标记。但是到目前为止，在HGP中尚未确定可靠的细胞表型。 我们最近通过使用高密度寡核苷酸微阵列（“ Genechips”）来表达谱图，通过表达谱图在HGPS细胞中显着上调了几个基因。这些基因现在可以作为HGP成纤维细胞独有的表型标记物。 为了检验HGP是常染色体显性突变的结果，将融合HGP和正常成纤维细胞的结果，并将通过微阵列分析杂化细胞的基因表达谱。这些实验还将确定可以在进一步研究中使用的标记，即HGPS成纤维细胞异常表达特征的基因在HGPS/正常杂交线中保持。 为了准备和测试可以在单细胞水平上测定异常表达基因的表达的细胞系，所选标记基因的启动子区域将与可通过荧光（例如绿色荧光蛋白或GFP）测定的报告基因融合，并稳定地转染到正常的纤维纤维中。将选择细胞系，其中GFP报告基因的表达完全反映了内源基因的表达。 为了克隆HGPS基因，由HGPS细胞制备的高滴度逆转录病毒cDNA文库将用于感染带有报告基因构建体的细胞系。感染细胞的池将通过流式细胞术和细胞分类来分析，以鉴定报告基因表达成为HGPS/正常杂种而不是正常细胞系的特征的细胞。 一旦以这种方式鉴定了假定的HGP基因，将在正常和HGPS细胞中确定基因的完整序列，以确定突变变化的性质。