生命科学学院知识库

生物体正是依赖于它自身基因表达在时间上和空间上的调控，才得以正常分化、发育、繁殖和调节代谢。在脊椎动物的内耳发育中，细胞分化命运的决定有赖于复杂的基因调节通路，此调节通路涉及 Wnt 家族。Axin2 是正规的 Wnt 调节通路中一个副调节因子，它通过促进 β-catenin 的降解，抑制信号的传导。我们发现了一个鸡的等位基因 cAxin2，于 HH-14+期，它初始表达在发育内耳的耳杯中，并在内耳的形成及进一步耳囊的分化过程中显示了一个动态的表达方式。
在这项研究中，我们通过应用活体电穿孔技术操控基因在耳囊中表达的方法，研究了用 RNA interference(RNAi)诱导靶基因沉默的可行性。我们的结果显示，应用增强的绿荧光蛋白、cAxin2 和短发夹 RNA 构建质粒通过活体电穿孔技术转染后，在耳囊中，可以看到很强的绿荧光信号。进一步的研究发现，用原位杂交技术检测基因cAxin2、Paired Box2 (cPax2)及 Bone Morphogenetic Protein 4(cBMP4)在转染后耳囊中的表达，显示 cAxin2 的表达被短发夹 RNA 抑制、cPax2 被下调而 cBMP4 被上调、并能看到一个异常增大的耳囊。结果提示，cAxin2 是一个内耳发育过程中非常重要的调控基因。

The decision of the cell fate specification in the developing vertebrate inner ear is dependent on complex regulatory genetic pathways involving in the Wnt families.
Axin2 is a negative regulator of the canonical Wnt pathway that suppresses signal transduction by promoting degradation of ß-Catenin. We have identified a chick
homolog gene，cAxin2，which is expressed in the otic cup beginning at stage HH14+ and exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In this study，we explored the possibility of using RNA interference（RNAi ） -induced targeted gene silencing introduced by in ovo electroporation to manipulate gene expression in the otic vesicle. We have
demonstrated that a strong enhanced green fluorescent protein (EGFP) signal can be detected in the otic vesicle after transfection with pEGFP-cAxin2-shRNA constructs by in ovo electroporation. The further investigation with in situ hybridization for genes cAxin2，Pared box2(cPax2)，and bone morphogenetic protein 4(cBMP4)show that the cAxin2 expression was inhibited by short hairpin RNA (shRNA)，cPax2 was down regulated and cBMP4 was up regulated as well as an abnormally enlarged  otic vesicle in the transfected otic vesicle. Our results suggest that cAxin2 is important molecular required for inner ear development.