| Project/Area Number |
07680570
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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 |
環境影響評価(含放射線生物学)
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| Research Institution | Tohoku University |
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Principal Investigator |
KIKUCHI Hideaki Institute of Development, Aging and Cancer Associate Prof., 加齢医学研究所, 助教授 (60006111)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUI Yasuhisa Institute of Development, Aging and Cancer Associate Prof., 加齢医学研究所, 助教授 (40241575)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Dioxin / Genomic imprinting / primordial germ cells / PCR / ゲノムプリンティング |
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| Research Abstract |
In order to determine the influence of dioxin on the next generations, we established the detection method to know the changing pattern of genomic imprinting. The basic theory is PCR amplification of the fragment which contains MspI site/HpaII site. MspI can cleave methylated sequences but HpaII can not. The test genomic DNA is digested with MspI or HpaII.The digested DNA was used as template for PCR.Only the undigested DNA with HpaII can produce amplified fragment. From calibration curve of amplified fragment, we can calculate the rate of methylation on the HpaII site. We selected H19 gene-site 9 (H19-9) on the mouse genome to check the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using embryonic germ (EG) cells as a model system of primordial germ (PG) cells, because DNA methylation is resetted during the early developmental stages of PG cells. H19 gene product is transcribed from the chromosome 7 of maternal origin. DBA-3-7 cells, one of the EG cells established by Matsui et al., were plated on the feeder cells of STO (3x106 cells/90cm dish), after 48h of plating the cells were treated with 2 nM or 20 nM of TCDD for 24 h or 48 h. At each time point, the cells were harvested and were used for DNA preparation. DNA methylation rate was determined by our methods and almost 100% of H19-9 site was methylated in TCDD treated and also untreated cells.
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