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Chicken embryos are used widely in the fields of developmental biology and neurobiology. The chicken embryo also serves as a model to analyze gene expression and function using in ovo electroporation. Plasmids may be injected into the spinal cord or tectum of the chicken central nervous system by microinjection for electroporation. Here, we developed a novel method that combines in ovo electroporation and neuronal culturing to study gene function in the chicken tectum during embryo development. Our method can be used to study in-vivo and in-vitro exogenous genes’ function. In addition, live cell imaging microscopy, immunostaining, and transfection can be used with our method to study neuronal growth, development, neurite growth and retraction, and axonal pathfinding. Our result showed that axons were present in isolated neurons after culturing for 24 h, and cell debris was low after replacing the media at 48 h. Many GFP-expressing neurons were observed in the cultured cells after 48 h. We successfully cultured the neurons for 3 weeks. Together, this method combines in ovo electroporation and neuronal culturing advantages and is more convenient for the gene function analysis.