MOUSE X CHROMOSOME INACTIVATION

小鼠 X 染色体失活

• 批准号: 2184366
• 负责人: Christine M. Disteche
• 金额: $ 20.38万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184366
• 关键词: alleles; embryogenesis; gene expression; genetic mapping; genetic strain; hybrid cells; in situ hybridization; nucleic acid sequence; polymerase chain reaction; sex chromosomes; sex linked trait; tissue /cell culture

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.

X染色体失活导致相同剂量的基因表达 在哺乳动物的雄性和女性之间。 但是，并非所有X连锁基因 如发现的发现 几种逃避人类失活的基因。 这是不知道的 逃脱X的基因在成年中是否从发作开始 胚胎发生中的失活。 特别感兴趣的是Xist基因 仅从非活动X染色体表达，可能会发挥 在X失活的开始中的作用。 在此提案中，我们概述了 在体内检查基因X灭活状态的实验 成人和胚胎小鼠。 我们计划分离出逃脱x失活的新小鼠基因 人X小鼠混合细胞系，仅保留无活跃的小鼠x 在选择性压力下，新霉素抗性基因的染色体 插入该染色体。 鼠标特异性的成绩单相对应 逃脱X灭活的基因将从cDNA文库中分离出来 由杂化细胞系构建。 然后，我们将通过原位映射 与小鼠染色体的杂交，从中分离出来的新基因 杂交细胞系和现有的X连锁基因已知可以逃脱x 人类失活。 除了在小鼠中定位基因之外， 分析可能揭示了Y同源物的存在。 我们将确定 通过利用a 鼠标X-小气体易位，其中正常X染色体不活跃 在所有细胞中以及小鼠物种之间的遗传变异以评估 通过逆转录酶在给定基因座处的等位基因表达 聚合酶链反应测定法。 我们将把这些研究扩展到 小鼠胚胎利用优先的父亲x- 胚外膜中的染色体灭活。 我们将遵循 Xist在胚胎中的表达，以查看其表达的发作是否 与该X灭活相关。 转基因的表达 以前显示出在成年小鼠中逃脱X失活的 在胚胎发生过程中紧随其后，以确定是否有重新激活 转基因或它是否从发病中逃脱。 最后，我们将在其他情况下寻找早期复制的区域 延迟复制的无活性小鼠X染色体，可能描述 染色体区域包含逃脱X失活的基因。