CORE--GENE EXPRESSION

（根据申请人摘要改写）提出设立该核心设施的主要原因是，SCOR提案中有多个研究者的项目需要对特定基因进行表达分析，以了解心脏的发育和心脏病中错误表达基因的发病机制（项目2、3、4、5）。 对基因进行功能解剖的关键因素之一是分析其时空表达模式。基因在发育组织中何时何地表达对于理解心脏发育和血管疾病的分子基础至关重要。 表达分析明确地和容易地通过使用选择的基因探针对整个小鼠胚胎和胚胎切片进行原位杂交来解决。 通过利用来自不同发育阶段的胚胎和各种胚胎切片，可以确定感兴趣的基因的时间和空间表达模式。 发育中的小鼠胚胎为研究基因表达提供了理想的模型。 人类发育缺陷的基因在小鼠中几乎总是有密切相关的同源物（1）。 此外，由于小鼠和人类基因之间的一级序列的广泛保守性，跨物种杂交通常是成功的。 考虑到获取人类胚胎样本的困难，小鼠提供了一个理想的替代品。 此外，基因的蛋白质产物的定位有助于在抗体可用的情况下进行分析。因此，将建立一个核心设施，储存整个小鼠胚胎和一系列发育阶段的胚胎切片，并使用候选基因探针和特异性抗体的免疫组织化学进行原位杂交实验和分析。整个胚胎和切片胚胎的原位杂交是复杂和耗时的，需要在小鼠饲养，胚胎分期，显微镜和分子生物学的各种技能（2）。 这种类型的研究非常适合由核心机构进行，并为具有不同特定目标但对心脏发育和/或疾病基因分析有共同兴趣的各种研究者提供服务。 这个核心的主要目的是为了促进基因的鉴定，参与圆锥动脉干畸形的遗传基础。 该核心将提供技术专长和设施，以进行空间表达研究，并协助不同专业的研究人员了解其心脏特异性基因的生物学。 项目2、3、4和5将能够利用这一核心进行基因表达分析。 表达研究需要各种各样的技能来建立。此外，由于费城儿童医院已经向奥凯博士提供了开始这一核心的种子资金，因此这一拟议的核心已经具备了开始提供服务所需的大部分设备和技术专长。

(Adapted from the Applicant's Abstract) The main reason for proposing to set up this core facility is that there are several investigators involved in the SCOR proposal whose projects require expression analysis of specific genes as part of understanding the development of the heart and pathogenesis of mis-expressed genes in heart disease (Projects 2, 3, 4 and 5). One of the crucial factors necessary for the functional dissection of a gene is the analysis of its spatial and temporal expression pattern. Where and when a gene is expressed in a developing tissue is essential to understanding the molecular basis of heart development and vascular disease. Expression analyses are addressed definitively and easily by in situ hybridization of whole mouse embryos and sections of embryos using a gene probe of choice. By utilizing embryos from different developmental stages and a variety of embryo sections, both the temporal and spatial expression patterns of genes of interest can be determined. The developing mouse embryo provides an ideal model in which to study gene expression. Genes responsible for developmental defects in humans almost always have a closely related homologue in mouse (1). Moreover, due to extensive conservation of primary sequence between mouse and human genes, cross species hybridizations are frequently successful. Given the difficulties with obtaining samples of human embryos, the mouse provides an ideal alternative. In addition, location of the protein product of a gene facilitates analysis where antibodies are available. Accordingly, a core facility will be established that will bank whole mouse embryos and embryo sections from a range of developmental stages and perform in situ hybridization experiments and analyses using candidate gene probes and immunohistochemistry with specific antibodies. In situ hybridization of whole and sectioned embryos is complex and time consuming and requires a variety of skills in mouse husbandry, embryo staging, microscopy and molecular biology (2). This type of study is ideally suited to being performed by a core facility and as a service to a variety of investigators with different specific goals but a common interest in developmental and/or disease gene analysis of the heart. The main aim of this core will be to facilitate the identification of genes involved in the genetic basis of conotruncal malformations. This core will provide the technical expertise and facilities to perform spatial expression studies and to assist investigators of varying specialties with the tools to understand the biology of their heart specific genes. Projects 2, 3, 4 and 5 will be in a position to utilize this core for gene expression analyses. Expression studies require a wide variety of skills to set up. Moreover, this proposed core already has most of the necessary equipment and technical expertise in place to begin providing a service since The Children's Hospital of Philadelphia has already provided Dr. Oakey with seed money to begin this core.