Development of assay system for in vivo promoter activity using homologous recombination method

同源重组法体内启动子活性测定系统的开发

基本信息

批准号：
10557240
负责人：
INOUE Ituro
金额：
$ 2.94万
依托单位：
Institute for Molecular and Cellular Regulation
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The molecular basis of single gene mendelian disorders resulting from gain or loss of function is being clarified at a rapid pace. Progress in the genetics of common disease, by contrast, has been frustratingly limited, as will be discussed by reference to essential hypertension. We have provided an indictment that the molecular variant in angiotensinogen gene could constitute susceptibility for essential hypertension. We have identified a molecular variant at -6 nucleotide position from the transcription start site in the core promoter in angiotensinogen gene. By the use of genetic association study, A(-6) allele is more frequently observed in hypertensive subjects than G(-6) allele. Functional analysis was performed by in vitro reporter assay using human hepatoma cells (HepG2), and we observed that A(-6) allele has higher transcriptional activity than G(-6) allele. However, in vitro analysis may not reflect the in vivo transcriptional activity. This dilemma needs to be cleared becaus … More e variation in the promoter region could become important in human common disorder.Homologous recombination is becoming a routine protocol for gene knock-out and gene replacement. We applied this method to study transcription activity in vivo. Mouse genomic angiotensinogen gene (15kb) was cloned from a library of 129 origin. The clone, containing 7kb promoter region, was subcloned into the vector (TT222) for targeting which was supplied from Mario Cappecci (Human Genetics, University of Utah). A fragment of the promoter was subcloned into pBluescript and PCR mutagenesis was performed to introduce a molecular variant in the promoter region. The fragment was again subcloned into TT222 targeting vector followed by transfection into mouse somatic cell lines. Using mouse hepatoma cell (Hepa 1-6) and mouse proximal tubular cell line (tsMPT), we could not obtain a clone, in which homologous recombination occurs. Probably due to low efficiency of homologous recombination in somatic cells, we are currently using ES cells for making recombinated cell line. Less

相比之下，由功能获得或丧失引起的单基因孟德尔疾病的分子基础正在迅速阐明，而常见疾病的遗传学进展却令人沮丧地有限，我们将参考原发性高血压进行讨论。已经提供了血管紧张素原基因中的分子变异可能构成原发性高血压的易感性的指控，我们通过利用遗传技术，在血管紧张素原基因核心启动子的转录起始位点的-6个核苷酸位置上鉴定了一个分子变异。关联研究中，A(-6) 等位基因在高血压受试者中比 G(-6) 等位基因更常见，通过使用人肝癌细胞 (HepG2) 的体外报告基因测定进行功能分析，我们观察到 A(-6) 等位基因。等位基因比 G(-6) 等位基因具有更高的转录活性，但是，体外分析可能无法反映体内转录活性，因为启动子区域的 e 变异可能在人类中变得很重要。同源重组是基因敲除和基因替换的常规方案，我们应用该方法从129个来源的文库中克隆了小鼠基因组血管紧张素原基因（15kb）。将启动子区域亚克隆到用于靶向的载体(TT222)中，该载体由Mario Cappecci (Human Genetics, University of Utah)提供。亚克隆到 pBluescript 中，并进行 PCR 诱变以在启动子区域引入分子变体。将该片段再次亚克隆到 TT222 靶向载体中，然后使用小鼠肝癌细胞 (Hepa 1-6) 和小鼠近端肾小管转染。细胞系（tsMPT），我们无法获得发生同源重组的克隆，可能是由于体细胞中同源重组的效率低。目前正在使用 ES 细胞来制备重组细胞系。