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

我们构建了携带含有Xist基因的YAC克隆的小鼠转基因系，以研究影响Xist表达和启动x -失活的因素。获得了两个转基因品系，一个在常染色体上携带4个拷贝，另一个在Y染色体上携带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，从y插入的转基因中观察到Xist的表达，其水平与正常雌性小鼠的水平相当。常染色体转基因Xist基因的5‘和3’甲基化位点是超甲基化的，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连锁的转基因，Xist基因的甲基化位点5‘和3’是低甲基化的，反映了在非活性X染色体上发现的模式。然而，在活性转基因位点上，5‘和3’甲基化水平已经解耦。由于β -geo报告基因已被修饰在YAC右臂上，我们在LacZ染色的基础上分析了β -geo在Tg15小鼠胚胎中的表达。单细胞期胚胎未被X-gal染色。2-4细胞期出现LacZ染色，桑葚胚期出现强染色。然而，胚泡期后，胚胎不染色。这些数据可能表明，在Xist附近的序列可以启动一些与x失活过程相关的现象。

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.