MANIPULATING MHC GENE EXPRESSION IN ES CELL CHIMERAS

本研究的总体目标是分析课堂的表达方式 I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 β2-微球蛋白，免疫学家现在认识到抗原肽 是I类分子的基本结构成分。我们最近 研究表明，需要的多肽转运体分子 MHC I类组装受发育调控。这样做的一个主要目的是 建议对MHC类别的组件的表达进行表征 MHC-I类分子表面表达所需的I型多肽装载机制 在早期胚胎中。原位杂交实验将分析 MHC编码转运蛋白表达的时空模式 分子和蛋白酶体成分。考察……的职能作用 在体内，我们将通过同源重组来破坏HAM1基因座 并将零突变引入生殖系。我们的 最近的实验证明MHC I类和Beta2m基因 在早期阶段表达不是协调调节的 植入后发育。事实上，我们发现次要的 滋养层巨细胞强烈表达MHC-I类，但未检测到 β2m基因。一个主要的目标是进一步描述个人的特征 I类转录本和确定哪些MHC I类基因产物是 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 杀伤力。进一步检测异位H2D-d的致畸作用(S) 在小鼠早期胚胎中的表达，我们将确定胚胎是否 致命性与I类分子的异常组装有关 和/或可归因于细胞内H链的过度表达。 我们还将检查MHC I类表达的后果(S) 特异性靶器官(S)使用组织特异性启动子。我们会 在AFP和mPLII启动子的控制下导入H-2D-d基因 直接表达到内脏卵黄囊的内胚层和继发性 滋养层巨细胞。我们是否应该努力获得 转基因菌株获得成功，下一个目标将是确定 H-2D-d链是否与Beta2m关联并输送到 细胞表面。如果转基因胚胎不能正常发育， 我们将研究MHC基因异位表达的可能性 这些胚胎外细胞谱系可能会触发母体免疫 针对异基因胎儿的反应。一个特别重要的问题 重点将是测试母体淋巴细胞是否渗入和破坏 不正常的胚胎。这些实验有望带来一个更清晰的 小鼠早期对MHC基因表达调控的认识 母胎相互作用中涉及的发育和过程。

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.