Buerli Thomas, Pellegrino Christophe, Baer Kristin, Lardi-Studler Barbara, Chudotvorova Ilona, Fritschy Jean-Marc, Medina Igor, Fuhrer Christian
Department of Neurochemistry, Brain Research Institute, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Nat Protoc. 2007;2(12):3090-101. doi: 10.1038/nprot.2007.445.
Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.
对原代神经元进行高效且持久的转染，是运用功能基因分析解决当前神经科学诸多问题的一项重要工具。神经元对细胞毒性敏感，采用大多数方法进行转染都很困难。我们提供了一种使用磁转染将cDNA和RNA干扰（短发夹RNA（shRNA））载体转染到体外培养数小时至21天的大鼠海马神经元（胚胎第18/19天）中的方案。该方案甚至允许将DNA双转染到一小部分海马神经元（γ-氨基丁酸能中间神经元）中，同时实现DNA和shRNA构建体的持久表达，且不干扰神经元分化。此方案使用廉价的设备和试剂，耗时1小时；利用混合海马培养物、一种转染试剂CombiMag和一块磁板；毒性低，适用于单细胞分析。我们三个实验室所做的修改也有详细说明。
