CARDIAC DEVELOPMENT DURING SITUS INVERSUS EMBRYOGENESIS

作为胚胎心脏发育的正常模式的一部分， 心脏通过心脏循环建立左右两极。 协调心电循环从而确立 哺乳动物的心脏是左侧的，目前还没有确定。一 获取有关这些因素的信息的策略是生成 以及改变心脏疾病模式的突变的特征 发展。我们已经制定了一种有效的策略来筛选 转基因小鼠的插入突变，最近已经 鉴定出一个有隐性突变的家系，该突变可导致坐位 反转。在这个家庭中，100%纯合的转基因小鼠 有一个镜像反转的左右两极 心脏以及包括肺在内的其他主要内脏器官， 肝、脾、胰腺和胃。新的突变并不是 先前在小鼠身上发现的iv突变的等位基因。自.以来 新的突变是插入突变，转基因插入 提供了一种可用于分离基因组的分子探针 失活基因所在的区域。 为了克隆新的反转部位基因并对其进行鉴定 它在心脏/胚胎发育调节中的作用 这项建议的具体目标是：1)建立染色体 新的倒位突变的位置和物理图谱；2) 确定该站点的结构和预测产品 转化基因，并通过靶向验证基因的功能 在ES细胞中的诱变；3)定义表达模式 反转部位基因产物，特别是在建立 心脏的左右两极，并表征了内部- 或编码蛋白的细胞外定位；以及4) 嵌合和双突变体心脏形态发生的研究 胚胎。因为突变基因在基因突变中起着至关重要的作用 定义正常心脏发育的两极， 编码蛋白的鉴定及其特性研究 它在胚胎发育过程中的表达模式将提供 关于一个协调因子的有价值的分子信息 心脏形态发生。

As part of the normal pattern of embryonic cardiac development, the heart establishes a left-right polarity through cardiac looping. The factor(s) that coordinate cardiac looping and thereby establish a left-sided heart in mammals have yet to be identified. One strategy to gain information about such factors is the generation and characterization of mutations that alter the patter of cardiac development. We have developed an efficient strategy to screen for insertional mutations in transgenic mice, and have recently identified a family with a recessive mutation that causes situs inversus. In this family, 100% of the homozygous transgenic mice have a mirror-image reversal of the left-right polarity of the heart as well as the other major internal organs, including lung, liver, spleen, pancreas and stomach. The new mutation is not allelic to the previously identified iv mutation in mice. Since the new mutation is an insertional mutation, the transgenic insert provides a molecular probe that can be used to isolate the genomic region where the inactivated gene is located. In order to clone the new situs inversus gene and the characterize its role in the regulation of cardiac/embryonic development, the specific aims of this proposal are to: 1) establish the chromosomal location and physical map of the new situs inversus mutation; 2) determine the structure and predicted product of this situs inversus gene and verify the function of the gene by targeted mutagenesis in ES cells; 3) define the pattern of expression of the situs inversus gene product, particularly during establishment of the left-right polarity of the heart, and characterize the intra- or extra-cellular localization of the encoded protein; and 4) investigate cardiac morphogenesis in chimeric and double mutant embryos. Since the mutated gene plays an essential role in defining the polarity of normal cardiac development, the identification of the encoded protein and the characterization of its pattern of expression during embryogenesis will provide valuable molecular information about a factor that coordinates cardiac morphogenesis.