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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。o -糖基化是一种常见的膜和分泌蛋白的翻译后修饰。丝氨酸(Ser)-和苏氨酸(Thr)-连接的低聚糖（o -聚糖）有四个常见亚型，称为核心1到4。o -聚糖的功能尚不清楚。特别注意的是核心20-聚糖在选择素介导的白细胞运输中的作用，但即使这些作用也没有完全定义。core1是由酶core1131,3 -半乳糖转移酶（C1GalT）形成的，是许多扩展型o -聚糖的前体，尤其是core2结构。利用Cre/loxP介导的基因靶向，我们开发了C1GalT全局性缺乏症小鼠（C1GalT -/-）和C1GalT基因两侧有loxP位点的小鼠（C1GalTta¿xed），这可用于制造条件性或组织特异性C1GalT缺乏症。C1GalT缺乏可导致胚胎出血和性交后14天死亡。我们建议使用我们的基因靶向小鼠，通过四个特定目的来研究体内核心1-基o -聚糖的功能：首先：我们将分析c1galt缺陷的胚胎，以确定出血是否是胚胎致死的原因，并确定出血是否由血管完整性缺陷、内皮细胞或造血细胞异常或凝血调节引起。其次，我们将通过将C1GalT与表达不同基因的转基因小鼠杂交，来培养具有组织特异性C1GalT缺陷的小鼠。第三，我们将通过凝集素和单抗染色以及使用外周糖苷酶和质谱分析的聚糖结构分析来检测野生型和C1GalTdeficient小鼠中o -聚糖的表达。第四，我们将评估C1GalT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.