| Project/Area Number |
63870002
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Department of Anatomy, School of Medicine, Keio University Shinanomachi-35, Shinjuku, Tokyo |
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Principal Investigator |
YASUDA Kenjiro Keio Univ. Anatomy Professor, 医学部, 教授 (90050327)
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| Co-Investigator(Kenkyū-buntansha) |
HIRAOKA Yoshiki Keio Univ. Anatomy Lecturer, 医学部, 助手 (80218768)
SHIOZAWA Masahide Keio Univ. Anatomy Lecturer, 医学部, 助手 (50170840)
AISO Sadakazu Keio Univ. Anatomy Assistant Professor, 医学部, 講師 (60138013)
YAMASHITA Shuji Keio Univ. Anatomy Assistant Professor, 医学部, 講師 (90050666)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | gamma-GTP / In situ hybridization / Northern blot analysis / In vitro transcription / γ-glutamyl transpeptidase / ノザンハイブリダイゼーション |
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| Research Abstract |
The in situ hybridization (ISH) has recently become a widely used procedure for detection and localization of nessanger RNA (mRNA) in cells or tissue section. To determine optimal condition for ISH, we performed Northern blot analysis by a cloned cDNA for human gamma-GTP during fetal development of liver.
1) Northern Blot Analysis gamma-GTP mRNA could be detected as early as at 12 weeks of gestation, and then increased gradually to a peak, approximately threefold of the amount at 12th week, at 40th week just before birth. The size of mRNA in fetal liver was 2.7kb and the mRNA of the saw size were detected both in human fetal kidney and human hepatocellular carcinoma as well as normal adult liver.
2) Probe Labeling To determine the method of probe labeling, we performed Northern blot analysis, with labeling of random priming system and SP6 system. The signal intensity of Northern blot analysis with SP6 system was superior to that with random priming system.
3) In Site Hybridization RNA probe were transcripted using biotinyl-dUTP derivative that contained an 11-atom spacer arm between the 5 position of the pyrimidine ring and the carboxyl group of the biotin moiety. When fetal liver were hybridized in situ with anti-sense strand gamma-GTP mRNA, the cytoplasm of the fetal hepatocytes were stained, and they were slightly hybridized with sense strand probe. Further study are needed to eliminate non specific staining from signals and to amplify the sensitivity of the reaction.
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