Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.
Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.
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DOI：
10.3791/675
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发表时间：
2008-02-13
期刊：
影响因子：
--
通讯作者：
Zito, Karen
中科院分区：
文献类型：
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作者：
Woods, Georgia;Zito, Karen
生物转染是一种用高速DNA包覆颗粒轰击组织转染细胞的物理方法。我们提供了大鼠海马切片生物转染的详细方案，从最初的DNA包覆子弹制备到最后使用基因枪进行器官型切片培养。基因枪转染是一种高效、简便的神经元转染方法，尤其适用于组织切片中一小部分细胞的荧光标记。在这个视频中，我们首先概述了用DNA涂覆金颗粒所需的步骤。我们接下来演示如何用金/DNA子弹在塑料管内部排列，以及如何切割该管以获得用于加载基因枪的塑料盒。最后，我们对大鼠海马切片进行生物转染，演示Bio-Rad Helios基因枪的处理，并提供故障排除建议，以获得健康和最佳转染的组织切片。
Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.