Biolistic transfection and expression analysis of acute cortical slices.
Biolistic transfection and expression analysis of acute cortical slices.
复制标题
急性皮质切片的基因枪转染和表达分析。
DOI：
10.1016/j.jneumeth.2020.108666
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发表时间：
2020
影响因子：
3
通讯作者：
Förster,Eckart
中科院分区：
文献类型：
--
作者：
Hamad,MohammadIK;Daoud,Solieman;Petrova,Petya;Rabaya,Obada;Jbara,Abdalrahim;Melliti,Nesrine;Stichmann,Sarah;Reiss,Gebhard;Herz,Joachim;Förster,Eckart
背景基因枪转染已被用于体外转染器官型培养物或分离培养物。在这里，我们修改了这项技术，使成功的转染急性脑切片，随后通过测量神经元的活动在几个hours.New methodWe建立了生物枪转染小鼠急性皮质切片来测量钙信号。将急性切片固定在血浆/凝血酶凝固物上并用钙传感器转染。成像可以在转染后4小时内进行，而不影响细胞violence.Results4小时后GCaMP 6s转染，急性切片显示显着的荧光蛋白表达水平，允许研究自发活动和受体药理学。虽然最佳气体压力（150 psi）和金颗粒尺寸（1 μm）证实了先前发表的方案，但发现5 μg DNA的量对于颗粒包被是最佳的。不需要长期表达转基因GECI的动物或OTC。在急性切片中，网络交互和连通性得以保留。该方法允许在功能性组织修饰可能生效之前4小时内获得生理读数。这种技术的局限性是随机转染，低表达效率时，使用特定的启动子，和排除或遗传操作，需要一个长时间的生理变化之前变得可测量的，如重组蛋白的表达，需要运输到遥远的亚细胞localization.ConclusionThe方法是最佳的短期调查的钙信号在急性切片。
BackgroundBiolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated culturesin vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours.New methodWe established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability.ResultsFour hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating.Comparison with existing methodsThe major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations.ConclusionThe method is optimal for short-time investigation of calcium signals in acute slices.