Xu Zhiyun, Liu Xin, Huang Xing, Yang Deqin
Hua Xi Kou Qiang Yi Xue Za Zhi. 2015 Aug;33(4):347-51. doi: 10.7518/hxkq.2015.04.005.
This study aims to investigate the expression of connexin 43 (cx43) gene during early development in zebra fish and provide a foundation for further research of cx43 gene in tooth development.
Total RNA was extracted within 72 h after fertilization of zebra fish embryos and then reversed transcribed to generate the cDNA library. The specific fragments of the cx43 gene were then cloned and connected to the PGEMT vector. After confirming the constructed plasmid, the corresponding RNA polymerase was chosen, and the digoxin-labeled anti-sense mRNA probe of cx43 was synthesized in vitro. The cx43 gene expression of zebra fish indifferent stages was carried out by in situ hybridization. The relationship of the cx43 gene expression and anatomy of the pharyngeal teeth were compared by alizarin red staining.
The mRNA antisense probe of cx43 was acquired. The positive signal of sepia was observed in the different stages of zebra fish pharyngeal teeth after fertilization. After fertilization for 9 days, the expression site of cx43 in situ hybridization was overlapped in accordance with the anatomical site of the pharyngeal teeth.
cx43 gene participates in tooth development and mineralization process and plays a crucial role in later mineralization.
本研究旨在探究斑马鱼早期发育过程中连接蛋白43(cx43)基因的表达情况,为进一步研究cx43基因在牙齿发育中的作用奠定基础。
在斑马鱼胚胎受精后72小时内提取总RNA,然后反转录生成cDNA文库。接着克隆cx43基因的特异性片段并连接到PGEMT载体上。确认构建的质粒后,选择相应的RNA聚合酶,体外合成地高辛标记的cx43反义mRNA探针。通过原位杂交检测斑马鱼不同阶段的cx43基因表达。用茜素红染色比较cx43基因表达与咽齿解剖结构的关系。
获得了cx43的mRNA反义探针。在斑马鱼受精后的不同阶段,在咽齿部位观察到深褐色阳性信号。受精9天后,cx43原位杂交的表达位点与咽齿的解剖位置重叠。
cx43基因参与牙齿发育和矿化过程,在后期矿化中起关键作用。