The screening of the genes that involved in the determination of the neural area and the neural induction

我们采用表达筛选策略对文库进行筛选，以确定调控神经外胚层发育的因子。我们从斑马鱼胚胎、小鼠胚胎和早期分化的小鼠胚胎干细胞中构建了质粒库。这些克隆被转化到大肠杆菌中，并被分成组，每组包含200个克隆。我们从每组中制备了质粒，并制作了用于显微注射的RNA。这些RNA被注射到斑马鱼胚胎的一到两个细胞期。用显微镜观察表型，用神经标记探针进行整体原位杂交。以Six3、ENG3、Krox20和Kheper为神经标记物。Kheper是我们克隆的新的转录因子。Kheper在整个神经外胚层中表达，并调控斑马鱼神经组织的发育。我们筛选了23×10^4个克隆，发现了许多众所周知的基因。此外，我们还成功地克隆了新的成纤维细胞生长因子家族基因、新的Bcl2家族基因和新的WNT家族基因。当这些基因在斑马鱼胚胎中过度表达时，神经外胚层就会扩大。新的成纤维细胞生长因子在中枢神经系统的前部有表达。提示新的成纤维细胞生长因子对前脑的发育有调节作用，我们将对这些因素进行上位性分析。我们想知道它们与Kheper的关系，以及它们如何调节神经组织的发育。

We screened cDNA libraries using expression screen strategy to identify the factors that regulated the development of the neuroectoderm. We made the plasmid cDNA libraries from zebrafish embryos, mouse embryos, and early differentiated mouse embryonic stem (ES) cells. These were transformed into the E.coli and divided in groups which contained 200 clones each. We prepared the plasmid from each groups and made RNAs for the microinjection. These RNAs were injected into the zebrafish embryos at one to two cell stage. We checked the phenotype by microscopic view and the whole mount in situ hybridization with neural marker probes. As the neural marker, we used six3,eng3,krox20 and kheper. kheper is the new transcription factor which we cloned. kheper is expressed in the whole neuroectoderm and regulated the development of the neural tissue of zebrafish.We screened 23 x 10^4 clones and found many well known genes. In addition, we succeeded to identify new FGF family gene and new Bcl2 family gene and new wnt family gene. When these are overexpressed in zebrafish embryos, the neuroectoderm were enlarged. The expression of the new fgf was detected in the anterior region of the central nervous system. It is suggested that the new FGF regulates the development of the forebrain.We are going to do the epistatic analysis of these factors. We would like to know the relationship of these with kheper and how they regulate the development of neural tissue.