ClC-7经NMD途径调控ClC-3的表达恢复颅颌面表型的效应和机制研究

遗传补偿效应（GCR）被认为是基因敲除和敲降之间表型差异的原因，但其机制却知之甚少。申请人前期研究发现：clcn7基因敲降斑马鱼出现了典型的颅颌面骨发育异常；clcn7基因敲除斑马鱼反而发育正常且其同源基因clcn3的表达显著升高；敲除斑马鱼中同时敲降无义突变mRNA降解（NMD）途径中upf3a后又出现了颅颌面骨异常，同时clcn3的表达恢复；并有文献提示NMD可参与特定基因的遗传补偿效应。基于以上我们提出“ClC-7经NMD途径调控ClC-3发生遗传补偿效应”的假说。本项目以基因敲除和敲降斑马鱼为模型，观察其颅颌面表型差异；确定差异表达同源基因并验证其功能; 解析触发遗传补偿效应的NMD途径关键分子；进一步通过基因沉默和过表达细胞等方法明确差异表达基因下游靶向的核心蛋白分子及功能。本项目为逆转伴发颅颌面骨发育异常的系列遗传性骨病提供新思路，为开展基于遗传补偿效应的基因治疗提供理论依据。

The genetic compensation response (GCR) has recently been proposed as a possible explanation for the phenotypic discrepancies between gene knockout and gene knockdown. However, the underlying molecular mechanism of the GCR remains uncharacterized. The applicant has previously found: the typical craniofacial dysplasia was found in clcn7 knockdown zebrafish, but the phenotype was not exhibit in clcn7 knockout zebrafish; the expression of homologous gene clcn3 was significantly increased in clcn7 knockout zebrafish, and the craniofacial dysplasia appeared again after upf3a knockdown. It is suggested that GCR of clcn7 knockout zebrafish occurs. In this project, using zebrafish knockdown and knockout models of clcn7 gene to observe the craniofacial phenotypic differences, and then to identify the differentially expressed homologous genes and verify their functions; to analyze the key molecules in NMD that trigger GCR; to identify the core protein molecules and functions targeted downstream of differentially expressed through gene over expression and silencing in cell. This project provides novel insights for the causation of a series of hereditary osteopathy associated with skull, maxillofacial bone dysplasia, and provides a theoretical basis for gene therapy based on GCR.