利用电穿孔技术将基因快速递送至有丝分裂后新皮层神经元体内。
In vivo rapid gene delivery into postmitotic neocortical neurons using iontoporation.
机构信息
Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland.
1] Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland. [2] Clinic of Neurology, Geneva University Hospital, Geneva, Switzerland.
出版信息
Nat Protoc. 2015 Jan;10(1):25-32. doi: 10.1038/nprot.2015.001. Epub 2014 Dec 4.
This protocol describes a method for directing the expression of genes of interest into postmitotic neocortical neurons in vivo. Microinjection of a DNA plasmid-amphiphilic molecule mix into the neocortex followed by delivery of an ad hoc electric pulse protocol during the first few days of life in mice allows rapid, focal and efficient expression of genes in postmitotic neurons. Compared with other gene delivery techniques such as in utero electroporation and viral infection, this method allows rapid (12 h), focal (50-200 μm), mosaic-like (50 to several hundred neurons) targeting of postmitotic neurons within existing circuits. This 'iontoporation' protocol, which can be completed within ∼20 min per mouse, allows straightforward assessment of genetic constructs in postmitotic cortical neurons and subsequent genetic, histological and physiological investigations of gene function.
本方案描述了一种在体将目的基因导入新生皮质神经元的方法。在新生鼠生命的最初几天，将 DNA 质粒-两亲分子混合物微注射到新皮层中，然后施加特定的电脉冲方案，可使处于有丝分裂后阶段的神经元快速、局灶、高效地表达基因。与其他基因传递技术（如胚胎电穿孔和病毒感染）相比，该方法可在现有的回路中快速（12 h）、局灶（50-200 μm）、类似镶嵌（50 到数百个神经元）地靶向处于有丝分裂后阶段的神经元。该“电穿孔”方案可在每只小鼠中约 20 分钟内完成，可直接评估处于有丝分裂后阶段的皮质神经元中的遗传构建体，并随后进行基因、组织学和生理学研究以探究基因功能。
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