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

我们构建了携带Xist基因的YAC克隆的小鼠转基因系，以研究影响Xist表达的因素和X失活的起始。获得了两个转基因品系--一个在常染色体上整合了4个拷贝，另一个在Y染色体上的一个单一位点整合了4个拷贝。在携带常染色体插入的小鼠中没有观察到Xist的表达。然而，在Y插入的转基因小鼠中观察到了Xist的表达，并且表达水平与正常雌性小鼠的水平相当。在常染色体转基因中，Xist基因的5‘和3’端的甲基化位点都是高甲基化的，似乎反映了在活跃的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5‘和3’位甲基化是在不活跃的X染色体上发现的低甲基化的反射模式。由于β-geo报告基因已被改造到YAC的右臂，因此我们在LacZ染色的基础上分析了β-geo在Tg15小鼠胚胎中的表达。单细胞期胚胎未见X-Gal染色。LacZ染色出现在2-4细胞期，桑椹胚期染色较强。然而，在囊胚期之后，胚胎没有被染色。这些数据可能表明，Xist附近的序列可以启动与X-失活过程相关的一些现象。

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.