BMC biotechnology · 2022
Efficient gene delivery into the embryonic chicken brain using neuron-specific promoters and in ovo electroporation.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Jung Kyung Min, Park Kyung Je, Kim Young Min, Han Jae Yong
Аннотация
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.