MANIPULATING MHC GENE EXPRESSION IN ES CELL CHIMERAS

本研究的总体目标是分析课堂表达 主要组织相容性复合体（MHC）的产物， 发育中的小鼠胚胎 除了MHC I类H链和 β 2-微球蛋白，免疫学家现在认识到，抗原肽 是I类分子的基本结构组分。 我们最近 研究表明， MHC I类组装是发育调节的。 其主要目的是 建议是表征MHC类组分的表达 MHC I类表面表达所需的I肽装载机器 早期胚胎 原位杂交实验将分析 MHC编码转运蛋白表达时空模式 分子和蛋白酶体组分。 审查的职能作用 HAM 1在体内，我们将通过同源重组破坏HAM 1基因座 并将无效突变引入生殖细胞。 我们 最近的实验表明，MHC I类和β 2 m基因， 表达在早期阶段不协调调节， 着床后发育。 事实上，我们发现次要的 滋养层巨细胞强烈表达MHC I类分子，但未检测到 β 2 m mRNA。 一个主要的目标是进一步描述个人 I类转录本，并确定哪些MHC I类基因产物是 表达。 此外，最近的研究表明， ES细胞嵌合体中的H-2D-d移植抗原导致产前 杀伤力 进一步检查异位H2 D-d的致畸作用 在早期小鼠胚胎中表达，我们将确定胚胎 致死性与I类分子的异常组装有关 和/或可归因于细胞内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 在AFP和mPLII启动子的控制下引入H-2D-d基因， 直接表达于内脏卵黄囊内胚层， 分别为滋养层巨细胞。 我们应该努力获得 转基因菌株成功，下一个目标将是确定 H-2D-d链是否与β 2 m相关并被转运至 细胞表面。 如果转基因胚胎不能发育到足月， 我们将研究异位MHC基因表达的可能性， 这些胚外细胞谱系可能会触发母体免疫 针对同种异体胎儿的反应。 一个特别重要 重点将是测试母体淋巴细胞是否浸润并破坏 异常胚胎 这些实验将有望导致一个更清晰的 对小鼠早期MHC基因表达调控的认识 参与母胎相互作用的发育和过程。

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.