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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 O-糖基化是一种常见的膜蛋白和分泌蛋白的翻译后修饰。丝氨酸(Ser)和苏氨酸(Thr)连接的寡糖(O-多糖)有四种常见的亚型，分别命名为核心1至4。O-多糖的功能尚不清楚。我们特别注意到 核心20-葡聚糖在选择素介导的白细胞转运中的作用，但即使是这些作用也没有完全确定。核心1是由核心1131，3-半乳糖基转移酶(C1GalT)形成的，是许多延伸的O-糖链的前体，特别是核心2结构。利用Cre/loxP介导的基因打靶，我们培育了C1GalT全球缺陷小鼠(C1GalT-/-)和C1GalT基因两侧带有loxP位点的小鼠(C1GalTta？xx)，这可以用来造成条件或组织特异性的C1GalT缺陷。C1GalT的全身性缺乏会导致胚胎出血和出生后14天(DPC)死亡。 我们建议使用我们的基因靶向小鼠，通过四个具体目标在体内研究基于核心1的O-糖链的功能：第一，我们将分析C1GalT缺陷的胚胎，以确定出血是否是胚胎死亡的原因，并确定出血是否源于血管完整性缺陷， 内皮细胞或造血细胞的异常，或凝血的调节。其次，我们将通过用不同的C1GalT转基因小鼠培育C1GalT基因缺陷小鼠来培育组织特异性C1GalT缺陷小鼠。第三，我们将通过凝集素和单抗染色检测O-糖链在野生型和C1GalT缺陷小鼠中的表达，并使用外切糖苷酶和质谱仪进行糖链结构分析。第四，我们将评估C1GalT在白细胞转运和淋巴细胞归巢中的意义。这些研究将阐明O-葡聚糖在发育、炎症、 体内的止血、免疫反应和其他生物功能。

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.