MANIPULATING MHC GENE EXPRESSION IN ES CELL CHIMERAS

操纵 ES 细胞嵌合体中的 MHC 基因表达

• 批准号: 3327186
• 负责人: ELIZABETH K BIKOFF
• 金额: $ 7.11万
• 依托单位: MOUNT SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3327186
• 关键词: cell transformation; early embryonic stage; embryo /fetus cell /tissue; embryo /fetus death; gene expression; genetic manipulation; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; glycoproteins; hematopoietic stem cells; histochemistry /cytochemistry; histocompatibility antigens; immunogenetics; in situ hybridization; laboratory mouse; leukocyte activation /transformation; major histocompatibility complex; membrane proteins; nucleic acid probes; pregnancy immunology; prenatal growth disorder; tissue mosaicism

The broad objective of this research is to analyse developmentally regulated expression of Class I products of the Major Histocompatability Complex in the mouse. In particular, the possibility that control of MHG gene expression may play a key role in maternal tolerance of the fetal allograft will be examined. We have designed a strategy that will allow us to study the consequences of ectopic expression of H-2D delta transplantation antigens in the developing embryo. A construct carrying the human Beta-actin gene promoter fused to the coding regions of the H-2D delta gene has been introduced into pluripotent embryonic stem (ES) cells. Although H-2D delta protein was not expressed at the cell surface in undifferentiated ES cells, significant amounts of H-2D delta membrane glycoproteins are detected on ES cells induced to differentiate in vitro. A major aim of the proposed investigation is to determine whether these transfected ES cell lines can contribute to all the somatic tissues when incorporated into normal embryos. Should our efforts to obtain live born chimeras be successful, the next goal will be to determine whether the introduced H-2D Delta gene can be transmitted in the germ line. Should transgenic chimeras fail to develop to term, we will describe the developmental failure and determine whether the abnormal phenotype (s) is attributable to defects in specific cell lineages. The spatial and temporal expression of Class I antigens in the developing embryos will be examined using immunohistochemical and in situ hybridization techniques. We will investigate the possibility that ectopic MHC gene expression in the extra- embryonic lineages may trigger a maternal immune response directed against the fetal allograft. A particularly important point will be to test whether maternal lymphocytes infiltrate and disrupt the abnormal embryos. These experiments will hopefully lead to a clearer understanding regulation of MHC gene expression during early mouse development and processes involved in maternal-fetal interactions.

这项研究的广泛目的是分析发展 主要组织兼容性I类产品的调节表达 在鼠标中复杂。特别是控制MHG的可能性 基因表达可能在胎儿的孕产妇耐受性中起关键作用 同种异体移植将检查。我们设计了一种策略，可以使我们 研究H-2D三角洲异位表达的后果 发育中的胚胎中的移植抗原。一个携带 人β-肌动蛋白基因启动子融合到H-2D的编码区域 Delta基因已被引入多能胚胎（ES）细胞中。 尽管H-2D三角洲蛋白在细胞表面未表达 未分化的ES细胞，大量的H-2D三角洲膜 在诱导分化体外的ES细胞上检测到糖蛋白。一个 拟议调查的主要目的是确定这些是否是否 当转染的ES细胞系可能有助于所有体细胞组织 掺入正常胚胎中。我们应该努力获得诞生 嵌合体成功，下一个目标是确定是否是否 引入的H-2D三角洲基因可以在种系中传播。应该 转基因嵌合体无法发展到学期，我们将描述 发育失败并确定异常表型是否为 归因于特定细胞谱系中的缺陷。空间和时间 将检查发育胚胎中I类抗原的表达 使用免疫组织化学和原位杂交技术。我们将 研究异位MHC基因表达在外部的可能性 胚胎谱系可能会触发针对的母体免疫反应 胎儿同种异体移植。一个特别重要的一点是测试是否 母体淋巴细胞浸润并破坏异常的胚胎。这些 实验将有望导致更清楚的了解 在早期小鼠发育和涉及过程中的MHC基因表达 在母亲互动中。