Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos.
Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos.
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DOI：
10.3791/53295
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发表时间：
2015-12-25
期刊：
影响因子：
--
通讯作者：
Wilson V
中科院分区：
文献类型：
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作者：
Huang Y;Wilkie R;Wilson V
早期小鼠胚胎的操作和培养是一种强大但在很大程度上未得到充分利用的技术，提高了该模型系统的价值。相反，细胞培养已广泛用于发育生物学研究。然而，重要的是确定体外培养的细胞是否真正代表体内细胞类型。将细胞移植到胚胎中，然后评估它们在发育过程中的贡献是确定体外培养细胞潜力的有用方法。在这项研究中，我们描述了一种方法，移植细胞到一个明确的网站早期植入后的小鼠胚胎，然后离体培养。我们还介绍了一种优化的电穿孔方法，该方法使用已知直径的玻璃毛细管，允许精确定位和调整接受外源DNA的细胞数量，同时具有高转染效率和低细胞死亡。这些技术，这不需要任何专门的设备，使实验操作的原肠胚和早期器官发生阶段的小鼠胚胎可能的，允许在培养的细胞亚群的承诺和原位遗传操作对细胞分化的影响进行分析。
Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.