FUNCTION OF GLI GENES IN SHH SIGNALING

刺猬（hh）信号通路与无脊椎动物和脊椎动物胚胎的形成有关。 果蝇的遗传分析表明，中断肘（ci）是信号通路的最下游成分。 在脊椎动物中有三个ci同源物，Gli1、Gli2和Gli3。 该提案的总体目标是通过使用敲入方法来确定这三个Gli基因在hh信号通路中的不同但重叠的功能。 具体而言，Gli2的内源性编码序列将被Gli1和Gli3的内源性编码序列取代。 该项目将分三步进行。 首先，分析Gli2基因组区域的5'区域以选择敲入位点。 同时，将制备Gli1和Gli3敲入构建体并在Sonic hedgehog应答细胞系MNS 70中进行测试。 第二步，将敲入构建体引入胚胎干细胞。 将产生嵌合胚胎和种系突变小鼠。 最后，将通过使用脊髓、肺和骨骼中的各种标记基因来分析与杂合和纯合突变小鼠相关的表型。对Gli基因功能的研究与人类发育和疾病直接相关，因为这三个基因的同源物存在于人类中，并且这些基因中的突变与癌症和发育缺陷有关。

The hedgehog (hh) signaling pathway is implicated in patterning both invertebrate and vertebrate embryos. Genetic analysis in Drosophila established that the cubitus interruptus (ci) is the most downstream component of the signaling pathway. There are three ci homologs in vertebrate, Gli1, Gli2 and Gli3. The overall goal of the proposal is to determine the distinct and yet overlapping function of these three Gli genes in hh signaling pathway by using the knock-in approach. Specifically, the endogenous coding sequence of Gli2 will be replaced by that of Gli1 and Gli3. This project will be done in three steps. First, the 5' region of Gli2 genomic region will be analyzed to select a knock-in site. In the meantime, Gli1 and Gli3 knock-in construct will be made and tested in Sonic hedgehog responsive cell line MNS70. In the second step, knock-in constructs will be introduced into embryonic stem cells. Chimeric embryos and germline mutant mice will be generated. Finally, the phenotypes associated with heterozygous and homozygous mutant mice will be analyzed by using various marker genes in the spinal cord, lung and skeleton. Studies on the function of Gli genes have direct relevance to human development and disease since homolog of these three genes exist in humans and mutations in these genes have been implicated in cancers and developmental defects.