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: 2199762
• 负责人: ELIZABETH K BIKOFF
• 金额: $ 27.13万
• 依托单位: HARVARD UNIVERSITY
• 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 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2199762
• 关键词: RNase protection assay; complementary DNA; developmental genetics; early embryonic stage; embryo /fetus death; embryo /fetus tissue /cell culture; gene expression; genetic manipulation; genetic transcription; genetically modified animals; hematopoietic stem cells; histocompatibility antigens; immunogenetics; in situ hybridization; laboratory mouse; major histocompatibility complex; molecular cloning; northern blottings; nucleic acid probes; pregnancy immunology; surface antigens; tissue mosaicism

The overall objective of this research is to analyze expression of class I products of the Major Histocompatibility Complex (MHC) in the developing mouse embryo. In addition to the MHC class I H chain and beta2-microglobulin, immunologists now appreciate that antigenic peptide is an essential structural component of the class I molecule. Our recent studies have shown that the peptide transporter molecules required for MHC class I assembly are developmentally regulated. A major aim of this proposal is to characterize expression of the components of the MHC class I peptide-loading machinery required for MHC class I surface expression in early embryos. In situ hybridization experiments will analyze the temporal and spatial pattern of expression of MHC-encoded transporter molecules and proteasome components. To examine the functional role of HAM1 in vivo, we will disrupt the HAM1 locus by homologous recombination in ES cells and introduce the null mutation into the germline. Our recent experiments demonstrated that MHC class I and beta2m gene expression is not coordinately regulated at early stages of post-implantation development. Indeed we found that the secondary trophoblast giant cells strongly express MHC class I but no detectable Beta2m mRNA. A major goal is to further characterize the individual class I transcripts and to determine which MHC class I gene products are expressed. In addition, recent studies indicate that over expression of H-2D-d transplantation antigens in ES cell chimeras results in prenatal lethalities. To further examine teratogenic effect(s) of ectopic H2D-d expression in early mouse embryos, we will determine whether embryonic lethalities are associated with aberrant assembly of class I molecules and/or can be attributed to over-expression of intracellular H chains. We will also examine the consequence(s) of MHC class I expression in specific target organ(s) using tissue-specific promoters. We will introduce the H-2D-d gene under control of the AFP and mPLII promoters to direct expression to the visceral yolk sac endoderm and secondary trophoblast giant cells respectively. Should our efforts to obtain transgenic strains be successful, the next goal will be to determine whether the H-2D-d chains are associated with beta2m and transported to the cell surface. Should the transgenic embryos fail to develop to term, we will investigate the possibility that ectopic MHC gene expression in these extra-embryonic cell lineages may trigger a maternal immune response directed against the allogeneic fetus. 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 of regulation of MHC gene expression during early mouse development and processes involved in maternal-fetal interactions.

这项研究的总体目的是分析类的表达 i主要组织相容性复合物（MHC）的产品 开发小鼠胚胎。 除了MHC I级链和 beta2-microglobolin，免疫学家现在感谢抗原肽 是I类分子的重要结构组成部分。 我们最近 研究表明，肽转运蛋白分子需要 MHC I类组装受发展的监管。 这是一个主要目的 建议是表征MHC类的成分的表达 I MHC I类表达式所需的肽加载机械 在早期的胚胎中。 原位杂交实验将分析 MHC编码转运蛋白表达的时间和空间模式 分子和蛋白酶体成分。 检查的功能作用 HAM1在体内，我们将通过同源重组破坏HAM1基因座 在ES细胞中，将无效突变引入种系。 我们的 最近的实验表明MHC I类和beta2m基因 表达在早期的早期不进行协调调节 植入后发展。 确实，我们发现次要 滋养细胞巨细胞强烈表达MHC I类，但无可检测到 beta2m mRNA。 一个主要目标是进一步描述个人 I类成绩单并确定哪些MHC I类基因产品是 表达。 此外，最近的研究表明，过度表达 ES细胞嵌合体中的H-2D-D移植抗原导致产前 致命性。 进一步检查异位H2D-D的致畸作用 在早期小鼠胚胎中的表达，我们将确定胚是否是否 致死性与I类分子的异常组装有关 和/或可以归因于细胞内H链的过表达。 我们还将研究MHC I类表达的后果 使用组织特异性启动子的特定靶器官。 我们将 将AFP和MPLII启动子控制的H-2D-D基因引入 直接表达对内脏蛋黄囊内胚层和次级 滋养细胞巨细胞分别。 我们应该努力获得 转基因菌株成功，下一个目标是确定 H-2D-D链是否与beta2m相关并运输到 细胞表面。 如果转基因胚胎无法发展到术语， 我们将研究异位MHC基因表达中的可能性 这些胚胎外细胞谱系可能会触发母体免疫 反应针对同种异体胎儿。 一个特别重要的 要点是测试母体淋巴细胞是否浸润和破坏 异常胚胎。 这些实验有望导致更清晰的 了解早期小鼠中MHC基因表达的调节 与母亲相互作用有关的发展和过程。