四足sal物种axolotl(Ambystomamexicanum)能够再生受伤的大脑。为了更好地理解大脑再生的机制,建立一种快速有效的功能获得和功能丧失方法来研究轴突大脑的基因功能是非常必要的。这里,我们建立并优化了一种基于电穿孔的方法,在端脑axolotl室管膜胶质细胞(EGC)中过表达或敲除/敲除靶基因.通过定位电极,我们能够在位于背侧的EGCs中实现EGFP的特异性表达,腹侧,中间,或侧脑室区。然后,我们通过电穿孔将Cdc42引入EGCs,研究了Cdc42在脑再生中的作用,其次是脑损伤。我们的发现表明,Cdc42在EGCs中的过表达并没有显着影响EGC的增殖和新生神经元的产生。但它破坏了它们的顶端极性,如ZO-1紧密连接标记的损失所示。这种破坏导致新生神经元的心室积累,它们未能迁移到它们可能成熟的神经元层,因此导致大脑再生表型延迟。此外,当将针对TnC(Tenascin-C)的CAS9-gRNA蛋白复合物电穿孔到大脑的EGC中时,我们实现了TnC的有效击倒。在电穿孔靶向区域,TnC表达在mRNA和蛋白质水平上都显著降低。总的来说,这项研究建立了一种快速有效的基于电穿孔的基因操作方法,从而可以研究axolotl脑再生过程中的基因功能。
The tetrapod salamander species
axolotl (Ambystoma mexicanum) is capable of regenerating injured brain. For better understanding the mechanisms of brain regeneration, it is very necessary to establish a rapid and efficient gain-of-function and loss-of-function approaches to study gene function in the
axolotl brain. Here, we establish and optimize an electroporation-based method to overexpress or knockout/knockdown target gene in ependymal glial cells (EGCs) in the
axolotl telencephalon. By orientating the electrodes, we were able to achieve specific expression of EGFP in EGCs located in dorsal, ventral, medial, or lateral ventricular zones. We then studied the role of Cdc42 in brain regeneration by introducing Cdc42 into EGCs through electroporation, followed by brain injury. Our findings showed that overexpression of Cdc42 in EGCs did not significantly affect EGC proliferation and production of newly born neurons, but it disrupted their apical polarity, as indicated by the loss of the ZO-1 tight junction marker. This disruption led to a ventricular accumulation of newly born neurons, which are failed to migrate into the neuronal layer where they could mature, thus resulted in a delayed brain regeneration phenotype. Furthermore, when electroporating CAS9-gRNA protein complexes against TnC (Tenascin-C) into EGCs of the brain, we achieved an efficient knockdown of TnC. In the electroporation-targeted area, TnC expression is dramatically reduced at both mRNA and protein levels. Overall, this study established a rapid and efficient electroporation-based gene manipulation approach allowing for investigation of gene function in the process of
axolotl brain regeneration.