COBRE: OK MED RES FOUND: P4: IN VIVO FUNCTIONS OF GALACTOSYLTRANSFERASE IN MICE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。O-糖基化是膜蛋白和分泌蛋白上常见的翻译后修饰。丝氨酸（Ser）和苏氨酸（Thr）连接的寡糖（O-聚糖）有四种常见的亚型，称为核心1至4。O-聚糖的功能还不清楚。核心20-聚糖在选择素介导的白细胞运输中的作用已受到特别关注，但即使是这些作用也没有完全确定。核心1由核心1131，3-半乳糖基转移酶（C1 GalT）形成，是许多延伸O-聚糖的前体，特别是核心2结构。使用Cre/loxP介导的基因靶向，我们已经开发了C1 GalT整体缺陷的小鼠（C1 GalT-/-）和C1 GalT基因侧翼为loxP位点的小鼠（C1 GalTtaixed），可用于创建条件性或组织特异性C1 GalT缺陷。C1 GalT的全面缺乏导致胚胎出血和死亡后14天（dpc）。我们建议使用我们的基因靶向小鼠研究核心1为基础的O-聚糖在体内的功能，通过四个具体的目标：第一：我们将分析C1 GalT缺陷的胚胎，以确定是否出血是胚胎致死的原因，并确定是否出血的结果从血管完整性缺陷，异常内皮细胞或造血细胞，或调节凝血。其次，我们将通过将C1 GalT突变小鼠与不同表达Cr的转基因小鼠交配来培育具有组织特异性C1 GalT缺陷的小鼠。第三，我们将研究O-聚糖在野生型和C1 GalT缺陷小鼠的表达凝集素和单克隆抗体染色和聚糖结构分析，使用外切糖苷酶和质谱。第四，我们将评估C1 GalT在白细胞运输和淋巴细胞归巢中的意义。这些研究将阐明O-聚糖在体内发育、炎症、止血、免疫反应和其他生物学功能中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. O-glycosylation is a common post-translational modification on membrane and secreted proteins. Serine (Ser)- and threonine (Thr)-linked oligosaccharides (O-glycans) have four common subtypes named cores 1 through 4. The functions of O-glycans are not well understood. Particular attention has been paid to the actions of core 20-glycans in selectin-mediated leukocyte trafficking, but even these roles are not completely defined. Core 1, which is formed by the enzyme core 1 131,3-galactosyltransferase (C1GalT), is the precursor for many extended O-glycans, especially the core 2 structure. Using Cre/loxP-mediated gene targeting, we have developed mice with a global deficiency of C1GalT (C1GalT -/-) and mice with the C1GalT gene flanked by loxP sites (C1GalTta¿xed), which can be used to create conditional or tissue-specific deficiencies of C1GalT. Global deficiency of C1GalT causes embryonic bleeding and death at 14 days post coitum (dpc). We propose to use our gene-targeted mice to study the functions of core 1-based O-glycans in vivo through four specific aims: First: We will analyze C1GalT-deficient embryos to determine if bleeding is the cause of embryonic lethality and to determine whether bleeding results from defects in vascular integrity, abnormalities in endothelial or hematopoietic cells, or regulation of blood coagulation. Second, we will develop mice with tissue-specific C1GalT deficiencies by breeding the C1GalT ta¿xedmice with different Creexpressing transgenic mice. Third, we will examine the expression of O-glycans in wild-type and C1GalTdeficient mice by lectin and mAb staining and by glycan structural analysis using exoglycosidases and mass spectrometry. Fourth, we will assess the significance of C1GalT in leukocyte trafficking and lymphocyte homing. These studies will elucidate the contributions of O-glycans in development, inflammation, hemostasis, immune responses, and other biological functions in vivo.