Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Korea.
BMC Biotechnol. 2022 Sep 2;22(1):25. doi: 10.1186/s12896-022-00756-4.
The chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging.
In the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters.
Transgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4.
We demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression.
鸡胚模型是探索神经和大脑发育潜在机制的一个有吸引力的系统，开发有效的遗传修饰技术对于在体内分析基因功能非常重要。尽管电穿孔和病毒载体介导的基因传递技术已被用于将外源 DNA 导入鸡胚胎细胞，但有效地、特异性地转导神经元仍然具有挑战性。
在本研究中，我们对普遍存在的 CMV 启动子和三个神经元特异性启动子（鸡钙/钙调蛋白依赖性激酶（cCaMKII）、鸡巢蛋白（cNestin）和人突触素 I）进行了比较研究。我们探索了使用选定的启动子通过胚胎内电穿孔来操纵鸡胚胎脑细胞中基因表达的可能性。
两个神经元特异性启动子（cCaMKII 和 cNestin）的转基因表达在体外培养的脑细胞中得到了初步验证，在体内，神经元特异性启动子驱动的 EGFP 转基因在脑细胞中的表达水平与普遍存在的 CMV 启动子相当或更高。由 cNestin 启动子驱动的 FOXP2 基因的过表达显著影响了靶基因 CNTNAP2 和 ELAVL4 的表达水平。
我们证明了通过含有神经元特异性启动子的构建体进行胚胎内电穿孔可以有效地将外源 DNA 引入活胚胎中的神经元细胞。胚胎内电穿孔为在胚胎发育过程中操纵基因表达提供了一种更简单、更有效的方法，这项技术将对神经元靶向转基因表达有用。
