原代大鼠海马神经元的高效转染

Efficient Transfection Method for Rat Primary Hippocampal Neurons

用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^(TM)与脂质体DOTAP和Lipofectaimine^(TM)的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^(TM)的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并用神经微丝(NF)抗体进行免疫细胞化学染色鉴定细胞类型。分别用DOTAP,Lipofectamine^(TM) and Nucleofector^(TM)包裹pCMV-eGFP质粒转染原代大鼠海马神经元。神经元的存活率用流式细胞促检测。实验结果表明:DOTAP和Lipofectamine^(TM)的基因转染效率仅为1.55％和2.45％,而Nucleofector^(TM)的转染效率则超过20％;细胞转染前后的存活率在DOTAP组分别为98.37％和88.35％,Lipofectamine^(TM)组分别为98.37％和90.11％,而在Nucleofector^(TM)组中分别为98.37％和51.82％。上述实验数据表明:Nucleofector^(TM)转染技术能高效并安全地转染原代大鼠海马神经元,但死亡率较高。

To compare Nucleofector^(TM), a new electroporation transfection method developed by Amaxa Biosystems, with two lipofection methods: DOTAP and Lipofectaimine^(TM) in El8 rat primary hippocampal neurons. The neurons freshly prepared by hippocampal dissection were identified with neuron-filament (NF) antibody by immunocytochemical staining. DOTAP, Lipofectamine^(TM) and rat neuron Nucleofector^(TM) were employed to transfer pCMV-eGFP plasmid into the rat hippocampal primary neurons respectively. The viability of the cells was assessed by fluorescence-activated cell sorting (FACS). The immunocytochemical staining for the neuronal marker NF showed that cells grown in Neurobasal^(TM) supplemented with B_(27), and then transfected, were mostly neurons. The transfection efficiency of DOTAP and Lipofectamine^(TM) reaches only 1.55% and 2.45% whereas Nucleofector^(TM) can achieve over 20% transfection efficiency. The viability of pre-and post-transfected cells with DOTAP is 98.37%/ 88.35% and 98.37%/90.11% with Lipofectamine^TM) whereas 98.37%/51.82% with Nucleofector^(TM). The results of the experiment showed that the newly developed Nucleofector^(TM) technology can lead a much higher transfection efficiency than other transfection methods in rat primary hippocampal neurons although the viability of transfected cells was somewhat reduced. Furthermore, this new technology is a suitable transfection method for most molecu- lar biological applications.