citations

Efficient Transfection Method for Rat Primary Hippocampal Neurons

Authors:

Miao HS, Yu LY, Lin B, Wu JC, Guo LH and Hui GZ

In:

Source: Chin J Cell Biol
Publication Date: (2004)
Issue: 26(3): 305-308

Research Area:

Neurobiology

Cells used in publication:

Neuron, hippo/cortical, rat: Species: rat; Tissue Origin: brain

Experiment

Efficient Transfection Method for Rat Primary Hippocampal Neurons MIAO Hong Sheng1,2, YU Lu Yang2, LIN Bo2, WU Jia Cai2, GUO Li He2*, HUI Guo Zhen1* （1Department of Neurosurgery, The First Hospital of Soochow University, Suzhou 215006, China; 2Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Shanghai 200031, China） Abstract: To compare Nucleofector™ a new electroporation transfection method developed by Amaxa Biosystems, with two lipofection methods: DOTAP and Lipofectaimine™ in E18 rat primary hippocampal neurons. The neurons freshly prepared by hippocampal dissection were identified with neuron-filament (NF) antibody by immunocytochemical staining. DOTAP, Lipofectamine™ and rat neuron Nucleofecto™ 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?SUP> supplemented with B27, and then transfected, were mostly neurons. The transfection efficiency of DOTAP and Lipofectamine™ reaches only 1.55% and 2.45% whereas Nucleofector™ 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™ whereas 98.37%/51.82% with Nucleofecto™ The results of the experiment showed that the newly developed Nucleofector™ 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 molecular biological applications. Key words: transfection; rat primary hippocampal neuron; DOTAP; lipofectamine™; nucleofector™; eGFP *Corresponding author, E-mail: lhguo@sibs.ac.cn

Abstract

To compare Nucleofector™ a new electroporation transfection method developed by Amaxa Biosystems, with two lipofection methods: DOTAP and Lipofectaimine™ in E18 rat primary hippocampal neurons. The neurons freshly prepared by hippocampal dissection were identified with neuron-filament (NF) antibody by immunocytochemical staining. DOTAP, Lipofectamine™ and rat neuron Nucleofecto™ 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 NeurobasalÜ? supplemented with B27, and then transfected, were mostly neurons. The transfection efficiency of DOTAP and Lipofectamine™ reaches only 1.55% and 2.45% whereas Nucleofector™ 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™ whereas 98.37%/51.82% with Nucleofecto™ The results of the experiment showed that the newly developed Nucleofector™ 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 molecular biological applications.