连接蛋白Cx50 S258F突变体对斑马鱼晶状体发育的影响

目的:研究与先天性白内障相关的连接蛋白Cx50 S258F突变体对斑马鱼晶状体发育的影响。方法:将野生型和携带S258F突变的Cx50编码区分别构建到pcDNA3.1myc/his真核表达载体上,通过体外转录获得相应的RNA产物,将RNA产物分别显微注射到1细胞期的斑马鱼胚胎,通过显微镜观察斑马鱼晶状体表型,并利用全胚原位杂交技术分析过表达野生型和突变型Cx50对晶状体标志基因Cx23表达的影响。结果:与野生型胚胎相比,S258F突变型Cx50过表达引起斑马鱼胚胎白内障表型,过表达野生型Cx50也引起白内障表型,但是畸形数量明显少于突变型,野生型和突变型Cx50的过表达均下调了Cx23的表达。结论:连接蛋白Cx50S258F突变体可引起斑马鱼白内障表型,提示Cx50的S258F突变体是造成先天性白内障的原因。

The effect of connexln 50 mutant S258F on zebrafish lens development

Objective： To explore the effect of missense mutation S258F of connexin 50 （Cx50） associated with autosomal dominant congenital cataract on zebrafish lens development. Methods： The fragment encoding wild-type or mutant-type was subcloned into pcDNA3.1myc-His. The 5＇ capped mRNA of both wild-type and mutant was synthesized from pcDNA3.1myc-His plasmids. A total of 50-100pg of RNA was injected into one-cell stage embryos. Living embryos were observed under a microscope. Whole-mount in situ hybridization on embryos were performed to detect the expression of lens marker Cx23. Results： Compared with wild-type embryos, overexpression of Cx50 S258F led to cataract formation in zebrafish lens. Although overexpressing wild-type Cx50 displayed similar phenotype, the percent of defect phenotype decreased significantly. Conclusion： Overexpressing Cx50 S258F leads to cataract formation in zebrafish lens, suggesting that the missense mutation S258F could be causative for congenital cataract formation.