|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)
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.
松居らにより樹立されたEG細胞は、始原生殖細胞のさまざまな発生段階の細胞の集団からなると考えられている。そこで、さまざまなゲノムインプリンティングを受けた状態のEG細胞があるので、マウス第7番の母親由来染色体から転写されるH19遺伝子DNAのメチル化状態を調べた。まずDAB-3-7細胞を、マイトマシン処理したフィーダー細胞STO(3x10^6 cells/90cm dish)上に蒔き、48時間後に終濃度2nMあるいは20nMとなるようにTCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin)を培地に加えた。TCDD処理後、24時間と48時間後に細胞を集め、DNA調製の材料とした。上記の方法に従い、各々のDNAのメチル化率を求めると、TCDD処理細胞も未処理細胞も、ほとんど100%メチル化されているという結果を得た。