Department of Cell Morphology and Molecular Neurobiology, Ruhr-University, Im Neuenheimer Feld 307, Bochum, Germany.
J Neurosci Methods. 2012 Aug 15;209(2):420-7. doi: 10.1016/j.jneumeth.2012.06.024. Epub 2012 Jun 29.
Cells of the central nervous system are notoriously difficult to transfect. This is not only true for neurons and glial cells but also for dividing neural stem and progenitor cells (NSCs). About ten years ago a major advance was provided by introduction of the nucleofection technology that allowed for transfection of approximately half of the exposed NSCs. However, limitations were encountered with the need for large numbers of NSCs for a single transfection and compromised survival rates with typically only one-third of the cells surviving the pulse conditions. Here, we report the establishment of a pulse protocol that targets NSCs with high efficiency and twofold higher NSC survival rates using the 4D Nucleofector device. We demonstrate that the established protocol not only provides a clear and significant improvement over existing protocols with transfection rates above 80% and two-thirds of the NSCs surviving for at least 48h, but also their unaltered differentiation along neuronal and glial lineages. This improved protocol for the transfection of sensitive mouse central nervous system derived cells will provide an important step forward for studies of gene function by overexpression or knock-down of genes in cultured NSCs.
中枢神经系统的细胞很难转染。这不仅对神经元和神经胶质细胞如此，对分裂的神经干细胞和祖细胞（NSC）也是如此。大约十年前，核转染技术的引入提供了一个重大进展，该技术可以转染大约一半暴露的 NSC。然而，需要大量的 NSC 进行单次转染，以及存活率受到影响，通常只有三分之一的细胞能在脉冲条件下存活，这限制了该技术的应用。在这里，我们报告了一种使用 4D 核转染仪的高效靶向 NSC 的脉冲方案，并提高了 NSC 的存活率，达到两倍以上。我们证明，该方案不仅在转染效率方面明显优于现有的方案（转染率超过 80%，三分之二的 NSC 至少能存活 48 小时），而且不会改变其向神经元和神经胶质谱系的分化。这种改进的用于敏感的小鼠中枢神经系统来源细胞的转染方案，将为在培养的 NSC 中过表达或敲低基因以研究基因功能提供重要的一步。
