Functional analysis of adrenocorticotropin receptor using the technique of gene targeting
| Project/Area Number |
10671028
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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 |
Endocrinology
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| Research Institution | Hokkaido University of Education |
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Principal Investigator |
KUBO Mitsumasa Hokkaido University of Education, Health Administration Center, Professor, 保健管理センター, 教授 (10205130)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMIZU Chikara Hokkaido University, Hokkaido University Medical Hospital, Instructor, 医学部附属病院, 助手 (00292029)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | adrenocorticotropin receptor / gene / knockout mouse |
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| Research Abstract |
Adrenocorticotropin (ACTH) induces steroidogenesis in the adrenal cortex. In addition, ACTH effects on adipose tissues and the central nervous system are also well known in rodents. ACTH receptor (ACTH-R) gene is a member of a melanocortin receptor gene family in which four other receptors for melanocyte-stimulating hormone are included. These receptors show sequence homology to each other and are co-expressed in some tissues, thereby ACTH-R function could not be distinguished from that of other melanocortin receptors. The aim of this study was to clarify the precise functions of ACTH-R, especially extra-adrenal functions, through the analysis of knockout mice lacking ACTH-R.We isolated phage clones containing ACTH-R gene from 129SVJ mouse genomic library. The targeting vector construct contained the diphteria toxin gene cassette, a 1.4kb DNA fragment as 5'-homology region, the PGK-Neo expression cassette (replaced whole coding region of ACTH-R gene), and a 5.5kb DNA fragment as 3'-homology region. After electroporation of the targeting plasmid into E14 cells, 500 surviving cell colonies in the presence of G418 were screened by Southern blotting. However, no clone with homologous recombination was obtained. This might be due to a short DNA fragment as 5'-homology region. Therefore, we constructed a targeting vector containing a 3kb DNA fragment as 5'-homology region, and transfetion of the vector construct into E14 cells is in process.
We could not generate mice lacking ACTH-R gene during the term of this study. Last year, mouse genomic sequence of chromosome 18 around ACTH-R gene (18p11.2) was appeared in GeneBank. It confirmed our results dealing with the genomic structure of ACTH-R we determined, and revealed a distance between first and fourth (coding) exon that was unclear before was 20kb.
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Report
(4 results)
Research Products
(6 results)
