Study of the effect of dioxin on the next generations using embryonic germ cells.

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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.