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首页> 外文期刊>Journal of Neuroscience Methods >Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.
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Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.

机译：聚乙烯亚胺提高有丝分裂后大鼠胎儿下丘脑神经元原代培养的转染效率。

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摘要

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNAratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.
机译：神经元原代培养物中基因调控序列的分析已被有效率的转染有报道质粒的有丝分裂后神经元所阻碍。我们描述了详细的条件，允许在血清补充的大鼠胎儿下丘脑细胞的原代培养中显着改善转染效率。转染的细胞在强大但非细胞特异性巨细胞病毒（CMV）启动子或促甲状腺激素释放激素（TRH）启动子的控制下表达绿色荧光蛋白（GFP），仅在TRH神经元中指导表达。使用CMV启动子-GFP质粒，我们测试了几种市售的转染试剂。聚乙烯亚胺（PEI）和Lipofectamine 2000可获得最佳结果。我们优化了PEI的转染程序，因为它可再现性更高。在35毫米培养皿中以10微克DNA，8.8的PEI / DNA比和6.9的PEI pH值以2.9 x 10（6）细胞的细胞密度转染细胞时，PEI的转染效果最佳。使用这些条件，我们能够在转染TRH启动子-GFP质粒后检测到GFP阳性神经元。 GFP阳性细胞已通过FACS成功纯化。这开辟了将哺乳动物CNS有丝分裂后神经元转染用于新应用的可能性，包括纯化特定神经元亚型。

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