| Project/Area Number |
10557240
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
Human genetics
|
|---|
| Research Institution | Institute for Molecular and Cellular Regulation |
|---|
Principal Investigator |
INOUE Ituro Gunma University Institute for Molecular and Cellular Regulation : Department of Cell Biology : Assoc. Professor, 生体調節研究所・調節機構部門, 助教授 (00192500)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TAKEDA Jun Gunma University Institute for Molecular and Cellular Regulation : Department of Cell Biology : Professor, 生体調節研究所・調節機構部門, 教授 (40270855)
持田 弘 (有)蛋白精製工業, 取締役
|
|---|
| Project Period (FY) |
1998 – 1999
|
| Project Status |
Completed (Fiscal Year 1999)
|
| Budget Amount *help |
¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1998: ¥2,900,000 (Direct Cost: ¥2,900,000)
|
|---|
| Keywords | Homologous recombination / Angiotensinogen / Promoter activity / 相同組み換え、 / 転写活性、 / 相同組換え / common disease |
|---|
| Research Abstract |
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
|
