Sp7/Osterix as a zinc finger transcription factor is expressed specifically in osteoblasts. Embryonic lethality of Sp7 knockout mice, however, has prevented from examining the functions of Sp7 in osteoblast and bone formation in live animals. Here we used TALEN, a versatile genome-editing tool, to generate one zebrafish sp7 mutant line. Homozygous sp7-/- mutant zebrafish are able to survive to adulthood. Alizarin Red staining and Micro-CT analysis showed that sp7-/- larvae and adult fish fail to develop normal opercula, and display curved tail fins and severe craniofacial malformation, while Alcian Blue staining showed no obvious cartilage defects in sp7-/- fish. Quantitative RT-PCR showed that a number of osteoblast markers including sppl, phex, collala, and collalb are significantly down-regulated in sp7-/- fish. Furthermore, col10a1a, whose ortholog is the cartilage marker in mice, was shown to be a novel downstream gene of Sp7 as an osteoblast marker in zebrafish. Together, these results suggest that Sp7 is required for zebrafish bone development and zebrafish sp7 mutants provide animal models for investigating novel aspects of bone development.
Pengfei NiuZhaomin ZhongMingyong WangGuodong HuangShuhao XuYi HOUYilin YanHan Wang
目的筛选肿瘤细胞中与生物钟分子振荡系统的重要组成部分PERl蛋白（period circadian protein homolog1）相互作用的蛋白质分子，为生物钟基因在肿瘤发生发展过程中的功能研究提供条件。方法应用细菌双杂交技术与人宫颈癌Hela细胞cDNA文库，以pBT为载体，构建pBT-PERl融合表达诱饵质粒，与pTRG连接的人宫颈癌HeIa细胞cDNA文库质粒共转化双杂交系统报告菌株，利用培养基的特殊选择性，筛选出阳性克隆并测序分析。结果筛选出14个蛋白编码基因，其中4个包含完整的蛋白编码序列，包括与线粒体动力学相关的OPA3蛋白，与铜代谢相关的CUTA蛋白，与细胞运动、定位等细胞事件相关的蛋白，与物质合成、代谢等生物化学反应相关的蛋白，还有在信号转导中发挥作用的相关蛋白。结论肿瘤细胞中与PER1蛋白存在潜在相互作用新蛋白的筛选和鉴定为研究人生物钟基因PERI在肿瘤发生发展中的功能提供了条件。
