猪的脊椎包括颈、胸、腰、荐和尾椎，其中颈、荐和尾椎相对固定而胸椎和腰椎存在变化。脊椎数与胴体长明显相关，每增加一根脊椎数可使成年猪体长增加15 mm。申请人及所在课题组通过一系列遗传学分析鉴别到了影响猪胸椎数的主效基因VRTN及其两个候选主效突变位点，但并未甄别出哪一个是真正的因果突变，有关该基因的时空表达特性、分子作用机理也仍属空白。本课题拟采用RT-PCR和Western blot研究VRTN基因在猪早期胚胎体节中的时空表达特性，通过Chip-seq分析其蛋白结合位点在胸椎形成调控网络中的作用；利用Luciferase报告基因表达体系和VRTN-LacZ转基因小鼠解析两个突变位点与基因转录调控的关联性并初步鉴别出因果突变位点；通过Crispr/cas9基因编辑技术将两个突变位点定点整合入小鼠VRTN基因中，通过基因编辑小鼠胸椎数是否增加以最终确定因果突变位点。

The porcine vertebrae consist of cervical, thoracic, lumbar, sacral and caudal vertebrae. The numbers of cervical and sacral vertebrae are always fixed whereas the number of thoracic and lumbar vertebrae varies considerably in pigs. The number of vertebrae is significantly associated with carcass length, and it has been estimated that one extra vertebra can expand the carcass length of about 15 mm in adult pigs. In our previous study, we characterize the VRTN gene that underlies phenotypic variation in thoracic vertebrae based on a series of genetic analyses. Moreover, we identified two most likely causative mutations in this gene. However, the real causative mutation is still unclear. And the temporal and spatial expression and molecular mechanisms of this gene remains unexplored. In this study, we plan to implement RT-PCR and Western blot to investigate the expression characteristics of VRTN in early embryonic body segment in pigs. Using the Chip-seq analysis, we attempt to dissect the roles of VRTN binding sites in regulation network of vertebra formation. In addition, the applicants intend to adopt Luciferase reporter assay and VRTN-LacZ transgenic mice to identify the association between these two mutations and VRTN transcriptional control, thus preliminarily distinguishing the causative mutation. Finally, we plan to introduce single and both mutations into mouse VRTN by site-directed integration using the Crispr/cas9 system, with the aim to finally determine the causative mutation affecting thoracic vertebrae on SSC7 that is expected to increase the number of thoracic vertebrae in genome-editing mice.