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

单基因孟德尔障碍的分子基础是由功能的获得或丧失引起的，目前正在迅速澄清。相比之下，常见病遗传学方面的进展却令人沮丧地有限，我们将在讨论原发性高血压时加以讨论。我们提出血管紧张素原基因的分子变异可能构成原发性高血压的易感性。我们在血管紧张素原基因核心启动子转录起始位点-6个核苷酸位置发现了一个分子变异。通过遗传关联研究发现，A（-6）等位基因在高血压患者中比G（-6）等位基因更常见。利用人肝癌细胞（HepG2）进行功能分析，我们发现A（-6）等位基因比G（-6）等位基因具有更高的转录活性。然而，体外分析可能不能反映体内的转录活性。这一困境需要解决，因为……启动子区域的更多变异可能在人类常见疾病中变得重要。同源重组正成为基因敲除和基因替换的常规方案。我们将这种方法应用于体内的转录活性研究。小鼠血管紧张素原基因组基因（15kb）从129个源文库中克隆。将含有7kb启动子区的克隆亚克隆到由Mario Cappecci （Human Genetics, University of Utah）提供的靶向载体TT222中。将该启动子片段亚克隆到pBluescript中，并进行PCR诱变，在启动子区域引入分子变异。将该片段再次亚克隆到TT222靶向载体上，转染小鼠体细胞。小鼠肝癌细胞（Hepa 1-6）和小鼠近端小管细胞系（tsMPT）均未获得同源重组的克隆。可能是由于体细胞同源重组效率较低，目前我们使用胚胎干细胞制造重组细胞系。少

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