MOUSE X-CHROMOSOME INACTIVATION

X染色体失活导致相同剂量的基因表达 雄性和雌性之间的差异。 然而，并不是所有的X连锁基因 最近的研究表明， 人类中几个逃避X失活的基因。 目前还不知道 在成人中逃避X失活的基因是否从一开始就这样做了， 在胚胎发生中的失活。 特别感兴趣的是XIST基因 这种基因只在不活跃的X染色体上表达， 在X染色体失活中的作用。 在这份提案中，我们概述了 实验，以检查在体内的X-失活状态的基因， 成年和胚胎小鼠。 我们计划分离新的小鼠基因，这些基因可以逃避X染色体失活， 人x鼠杂交细胞系，只保留了无活性的鼠X染色体 新霉素抗性基因的选择压力下的染色体 插入到那个染色体中。 小鼠特异性转录本 逃避X失活的基因将从cDNA文库中分离出来 由杂交细胞系构建。 然后我们将绘制地图， 杂交小鼠染色体，新的基因分离出的 杂交细胞系和已知逃避X染色体的现有X连锁基因 在人体内失活。 除了在小鼠中定位基因， 分析可以揭示Y同源物的存在。 康贝特人将以 成年小鼠体内基因的失活状态，通过利用 小鼠X-常染色体易位，其中正常X染色体失活 在所有细胞和小鼠物种之间的遗传变异， 通过逆转录酶在给定位点的等位基因表达 聚合酶链反应测定。 我们将把这些研究扩展到 小鼠胚胎，利用优先父系X- 胚外膜染色体失活。 我们将按照 Xist在胚胎中的表达，以观察其表达的开始是否 与X染色体失活有关 转基因的表达 先前显示在成年小鼠中逃避X失活的基因将被 在胚胎发生过程中进行跟踪，以确定是否有重新激活 或者它是否从一开始就逃脱了失活。 最后，我们将寻找早期复制的区域， 晚期复制的无活性小鼠X染色体， 含有逃避X失活的基因的染色体区域。

X-chromosome inactivation results in the same dosage of gene expression between males and females of mammals. However, not all X-linked genes are subject to X inactivation as recently shown by the finding of several genes that escape X inactivation in human. It is not known whether genes that escape X inactivation in adult do so from the onset of inactivation in embryogenesis. Of special interest is the XIST gene that is expressed only from the inactive X chromosome and may play a role in the onset of X inactivation. In this proposal, we outline experiments to examine in vivo the X-inactivation status of genes in adult and embryo mice. We plan to isolate new mouse genes that escape X inactivation from a human x mouse hybrid cell line that retains only the inactive mouse X chromosome under selective pressure for the neomycin-resistance gene inserted in that chromosome. Mouse-specific transcripts corresponding to genes that escape X inactivation will be isolated from a cDNA library constructed from the hybrid cell line. We will then map, by in situ hybridization to mouse chromosomes, the new genes isolated from the hybrid cell line and existing X-linked genes known to escape X inactivation in human. In addition to locating the genes in mouse, this analysis may reveal the presence of Y homologs. We will determine the inactivation status of the genes in adult mice in vivo, by exploiting a mouse X-autosome translocation where the normal X chromosome is inactive in all cells and the genetic variation between mouse species to evaluate allelic expression at a given locus by a reverse transcriptase polymerase chain reaction assay. We will extend these studies to the mouse embryo by taking advantage of the preferential paternal X- chromosome inactivation in extraembryonic membranes. We will follow the expression of Xist in embryos to see whether the onset of its expression correlates with that X inactivation. The expression of a transgene previously shown to escape X inactivation in adult mouse will be followed during embryogenesis to determine whether there is reactivation of the transgene or whether it escapes inactivation from the onset. Finally, we will look for regions of early replication, in the otherwise late-replicating inactive mouse X chromosome, that may delineate chromosomal regions that contain genes that escape X inactivation.