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Molecular mechanism of initiation of X inactivation using Xist YAC transgenic mouse

Xist YAC转基因小鼠启动X失活的分子机制

基本信息

批准号：
08672597
负责人：
MATSUURA Shinya
金额：
$ 1.41万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672597/
关键词：
Model mouse Xist X inactivation Counting mechanism transgenic mouse microinjection

项目摘要

We have constructed mouse transgenic lines carrying a YAC clone encompassing the Xist gene in order to investigate the factors influencing Xist expression and the initiation of X-inactivation. Two transgenic lines were derived - one carrying 4 copies integrated at an autosomal site and a second line carrying 4 copies integrated at a single site on the Y chromosome. Xist expression was not observed in mice carrying the autosomal insertion. However, Xist expression from the Y-inserted transgenes was observed and at levels commensurate with that found in normal female mice. Methylation sites in the autosomal transgene both 5' and 3' of the Xist gene are hyjpermethylated and appear to reflect methylation patterns observed on the active X chromosome. For the Y-linked transgene, methylation sites 5' and 3' of the Xist gene are hypomethylated feflecting patterns found on the inactive X chromosome. However, the 5' and 3' methylation levels have been decoupled at the active transgenic locus.As beta-geo reporter gene had been retrofitted on the YAC right arm, we have then analyzed beta-geo expression in Tg15 mouse embryos on the basis of LacZ staining. One cell stage embryos were not stained with X-gal. LacZ staining appeared at 2-4 cells stage, and strong staining was detected at morula stage. However, after blastcyst stage, embryos were not stained. The data might suggest that sequences in the vicinity of Xist can initiate some phenomenon associated with the process of X-inactivation.

我们已经构建了小鼠转基因系携带YAC克隆包含Xist基因，以研究影响Xist表达和启动X-失活的因素。得到两个转基因系-一个携带在常染色体位点整合的4个拷贝，第二个系携带在Y染色体上的单个位点整合的4个拷贝。在携带常染色体插入的小鼠中未观察到Xist表达。然而，观察到来自Y插入的转基因的Xist表达，并且其水平与在正常雌性小鼠中发现的水平相当。Xist基因的5'和3'常染色体转基因中的甲基化位点是超甲基化的，并且似乎反映了在活性X染色体上观察到的甲基化模式。对于Y连锁转基因，Xist基因的甲基化位点5'和3'是在失活X染色体上发现的低甲基化的表达模式。由于在YAC右臂上改造了β-geo报告基因，我们在LacZ染色的基础上分析了Tg 15小鼠胚胎中β-geo的表达。一细胞期胚胎未被X-gal染色。LacZ染色出现在2-4细胞期，桑椹胚期检测到强染色。囊胚期以后，胚胎不染色。这些数据可能表明，Xist附近的序列可以引发一些与X失活过程相关的现象。