We focused on the cartilage differentiation and regulation by tissue-specific transcription factors during the period of this Grant-in-Aid for Scientific Research

1.软骨组织特异性转录因子候选基因的筛选我们采用西南向筛选技术筛选了软骨细胞表达文库，筛选出与II型胶原基因增强子元件相互作用的基因克隆。4个阳性克隆(软骨特异性增强子结合蛋白CSEBP)进一步用Northernblotting和/或RT-PCR分析其mRNA的表达模式。这些克隆在软骨和脑等其他组织中表达，并在小鼠胚胎中表达。其中一个克隆是II型胶原C-前肽的cDNA2。为了研究作为II型胶原基因增强子结合蛋白的II型胶原C-前肽在体内的功能，我们构建了带有II型胶原启动子/增强子的II型胶原C-前肽的转基因小鼠，以实现该结构…的高效表达在软骨组织中，这些小鼠表现为轻度侏儒，长骨缩短，钙化延迟。免疫组织化学分析和免疫电子显微镜显示，该结构在生长板肥大软骨细胞外基质和肥大软骨细胞中均有表达。此外，在肥大软骨细胞核中检测到这些转基因分子，结合我们之前的体外研究，这些结果表明，II型胶原C-前肽可能被肥大软骨细胞摄取，转位到细胞核，并结合到II型胶原基因增强子的增强子区域，作为负反馈调节因子减少该基因的转录。为分析CSEBPs基因的体内功能，我们筛选了其中一个克隆的小鼠基因组文库，并构建了其中一个克隆的敲除载体。CSEBP-2基因有14个外显子，全长8kb，并在ES细胞中表达。我们已经构建了无启动子的结构，以产生CSEBP-2的敲除小鼠。较少

1. cDNA screening for candidate gene of tissue-specific transcription factors of cartilageWe have screened rhondrocyte cDNA expression library by South-western technique to identify cDNA clones which can interact with enhancer elements of type II collagen gene. Four positive clones (CSEBP ; cartilage specific enhancer binding protein) were further analyzed for their mRNA expressioin pattern by Nothern blotting and/or RT-PCR analysis. These clones were expressed in cartilage ant other tissues such as brain and hears in mouse embryos. One of these clones were cDNA for type II collagen C-propeptide.2. Analysis of transgenic mouse overexpressing type II collagen C-propeptide.In order to educidate in vivo function of type II collagen C-propeptide which was isolated as a binding protein of type II collagen gene enhancer, we have generated transgenic mice harboritig cDNA for type II collagen C-propeptide with type II collagen promoter/enhancer to achieve high level expression of this construc … More t in cartilage tissue These mice showed mild dwarfism with shortening of long bone and delayed calcification. Immunohistochemical analysis and immuno-electron microscopy demonstrated the expression of this construct in the extracelular matrix of hypertrophic cartilage in growth plate and in rER in hypersrophic chondrocyte. Furthermore, these transgene molecule was detectable in nucleus of hypertrophic cartilage.Taken together with our previous in vitro study, these results indicate the possibility that type II collagen C-propeptide was taken up hypertrophic chondrocyte, translocated to nucleus and bound to the enhancer region of type II collagen gene enhancer to reduce transcription of this gene as a negative feedback regulator.3. Analysis of gene structure and production of knock-out mice of CSEBPTo analyze in vivo function of CSEBPs, we have screened mouse genomic library and constructed knock-out vector for one of these clones. CSEBP-2 has 14 exons expanding 8 Kb and this gene was expressed in ES cells. We have generated promoterless construct to generate knock-out mice for CSEBP-2. Less