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

使用胚胎生殖细胞研究二恶英对下一代的影响。

基本信息

批准号：
07680570
负责人：
KIKUCHI Hideaki
金额：
$ 1.47万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680570/
关键词：
Dioxin Genomic imprinting primordial germ cells PCR ゲノムプリンティング

项目摘要

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.

为了确定二恶英对下一代的影响，我们建立了检测方法来了解基因组印记的变化模式。其基本原理是PCR扩增含有MspI位点/HpaII位点的片段。MspI能切割甲基化序列，而HpaII不能。用MspI或HpaII酶切待测基因组DNA，以酶切后的DNA为模板进行PCR，只有未用HpaII酶切的DNA才能产生扩增片段。从扩增片段的校准曲线，我们可以计算HpaII位点上的甲基化率。我们选择了小鼠基因组上的H19基因位点9（H19-9），以胚胎生殖细胞（EG）作为原始生殖细胞（PG）的模型系统来检查2，3，7，8-四氯二苯并-p-二恶英（TCDD）的影响，因为DNA甲基化在PG细胞的早期发育阶段被重置。H19基因产物转录自母体来源的7号染色体。DBA-3-7细胞，由Matsui等建立的EG细胞之一，接种于STO的饲养细胞上（3 × 106个细胞/90 cm培养皿），接种48小时后，用2nM或20 nM的TCDD处理细胞24小时或48小时。在每个时间点，收获细胞并用于DNA制备。用我们的方法测定DNA甲基化率，在TCDD处理和未处理的细胞中几乎100%的H19-9位点被甲基化。