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Functional analysis of adrenocorticotropin receptor using the technique of gene targeting

Research Project

Project/Area Number 10671028
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Endocrinology
Research InstitutionHokkaido University of Education

Principal Investigator

KUBO Mitsumasa  Hokkaido University of Education, Health Administration Center, Professor, 保健管理センター, 教授 (10205130)

Co-Investigator(Kenkyū-buntansha) SHIMIZU Chikara  Hokkaido University, Hokkaido University Medical Hospital, Instructor, 医学部附属病院, 助手 (00292029)
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
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)
Keywordsadrenocorticotropin receptor / gene / knockout mouse
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.

Report

(4 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Mitsumasa Kubo: "Alternate Exon in the 5'-Untranslated Region of the Human ACTH Receptor Gene."Endocrine Journal. vol.47. 543-548 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Chikara Shimizu: "Involvement of the Cholinergic Pathway in the Pathogenesis of Pituitary Cushing's Syndrome."Endocrine Journal. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kubo M., Shimizu C., Kijima H., Ishizuka T., Takano K., Takano A., and Koike T.: "Alternate exon in the 5'-untranslated region of the human ACTH receptor gene."Endocrine J.. 47. 543-548 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shimizu C., Yamane Y., Ishizuka T., Kijima H., Takano K., Takano A., Kubo M., and Koike T.: "Involvement of the cholinergic pathway in the pathogenesis of pituitary Cushing's syndrome."Endocrine J.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Mitsumasa Kubo: "Alternate Exon in the 5'-Untranslated Region of the Human ACTH Receptor Gene."Endocrine Journal. vol.47. 543-548 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Chikara Shimizu: "Involvement of the Cholinergic Pathway in the Pathoqenesis of Pituitary Cushing's Syndrome."Endocrine Journal. (in press). (2001)

    • Related Report
      2000 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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