| Project/Area Number |
17591113
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | KEIO UNIVERSITY |
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Principal Investigator |
HASEGAWA Tomonobu Keio University, School of Medicine, Associate Professor, 医学部, 助教授 (20189533)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | StAR / immortalized adrenal steroidogenic cell line / StAR knockout mice / 不死化副腎皮質ステロイ / トランスジェニックマウス |
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| Research Abstract |
The aim of this study was to elucidate the structure-function relationship of mouse steroidogenic acute regulatory protein (StAR) gene in steroidogenic cell line in vitro.
1.We established two types of immortalized adrenal steroidogenic cell lines ; one from wild type mice (wild cell line) and the other from StAR knockout mice (StAR (-) cell line), using temperature sensitive SV40 large T antigen transgenic mice.
2.We transfected wild type StAR gene into StAR (-) cell line by calcium phosphate method. We then confirmed the expression of wild StAR mRNA by RT-PCR.
3.We generated five mutated StAR genes by mutagenesis ; (1)one without mitochondrial targeting sequence, (2)one without last 28 amino acid, which is the identical mutant most commonly found in human disease due to StAR gene mutation, (3)one with S57A (putative phosphorylation site was substituted from serine to alamine), (4)one with S196A (another putative phosphorylation site was substituted from serine to alamine), and (5)one with both S57A and S196A. Each mutated StAR gene was introduced into expression vector.
4.We are transfecting these mutant StAR gene into Star (-) cell line by calcium phosphate method.
5.After confirming the expression of each mutated StAR gene, we will measure the adrenal steroid hormone in conditioned media from StAR (-) cell line, into which either wild type or each mutant StAR gene was transfected.
These studies will highlight the pivotal functional sites in StAR gene structure.
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