MOUSE X CHROMOSOME INACTIVATION

X染色体失活会导致相同剂量的基因表达 哺乳动物的雄性和雌性之间。然而，并不是所有的X连锁基因 如最近发现的那样，受到X失活的影响 人类中几个逃脱X失活的基因。目前尚不清楚 在成人中逃脱X失活的基因是否从一开始就这样 胚胎发育过程中的失活。特别有趣的是XIST基因 它只在不活跃的X染色体上表达，并可能起到 在X失活发生中的作用。在这份提案中，我们概述了 体内检测基因X失活状态的实验 成年和胚胎小鼠。 我们计划分离新的小鼠基因，从X失活中逃脱 只保留非活性小鼠X的人X鼠杂交细胞系 新霉素抗性基因在选择压力下的染色体定位 插入到该染色体中。相应的小鼠特定转录产物 逃脱X失活的TO基因将从cDNA文库中分离出来 由杂交细胞系构建而成。然后我们将通过现场绘制地图 与小鼠染色体杂交，从小鼠染色体中分离出新基因 杂交细胞系和已知逃逸X的现有X连锁基因 在人类体内失活。除了定位小鼠的基因外，这项研究 分析可能揭示Y同源基因的存在。我们将确定 体内成年小鼠体内基因失活状态的研究 正常X染色体不活跃的小鼠X常染色体易位 对所有细胞和小鼠物种间的遗传变异进行评估 逆转录酶在特定基因座上的等位基因表达 聚合酶链式反应分析。我们会把这些研究扩展至 小鼠胚胎利用父本的优先X- 胚胎外膜中的染色体失活。我们将遵循 观察Xist在胚胎中的表达是否开始表达 与X失活有关。转基因植物的表达 先前显示的逃脱X失活的成年小鼠将是 在胚胎发育过程中进行跟踪，以确定是否存在重新激活 或者它是否从一开始就逃脱了失活。 最后，我们将在其他方面寻找早期复制的区域 晚期复制不活跃的小鼠X染色体，可能描绘出 含有逃脱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.