Functional Cloning of Hutchinson-Gliford progeria gene

描述(由申请人提供)：沃纳综合征的基因，一种成人起病的孕激素综合征，最近通过位置克隆被鉴定出来。这种方法不能用于克隆哈钦森-吉尔福德早衰症(HGPS)基因，因为没有HGPS家系。因此，建议通过功能互补的方法克隆HGPS基因。要通过功能互补来克隆基因，关键是要在疾病细胞中识别一个可靠的、可测量的、正常细胞中不存在的表型标记。但到目前为止，还没有在HGPS中鉴定出可靠的细胞表型。我们最近通过使用高密度寡核苷酸微阵列(“GeneChips”)的表达谱鉴定了几个基因在HGPS细胞中的表达显著上调。这些基因现在可以作为HGPS成纤维细胞独有的表型标记。 为了验证被广泛接受的假设，即HGPS是常染色体显性突变的结果，将HGPS与正常成纤维细胞融合，并将通过微阵列分析杂交细胞的基因表达谱。这些实验还将确定那些可以用于进一步研究的标记，即那些在HGPS/正常杂交系中保持HGPS成纤维细胞异常表达特征的基因。为了制备和测试能够在单细胞水平上检测异常表达基因表达的细胞系，将所选择的标记基因的启动子区域与可用荧光分析的报告基因(如绿色荧光蛋白或GFP)融合，并稳定地转染正常成纤维细胞。将选择绿色荧光蛋白报告基因(S)表达与内源基因(S)表达完全一致的细胞系。为了克隆HGPS基因，从HGPS细胞中制备的高滴度逆转录病毒文库将被用来感染携带报告载体的细胞系。感染细胞池将通过流式细胞仪和细胞分类进行分析，以确定报告基因表达成为HGPS/正常杂交而不是正常细胞系的特征的细胞。一旦以这种方式确定了假定的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。

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.