Swezey R R, Epel D
Hopkins Marine Station, Stanford University, Pacific Grove, California 93950.
Cell Regul. 1989 Nov;1(1):65-74. doi: 10.1091/mbc.1.1.65.
We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.
我们描述了一种简单的电穿孔程序，用于在接近90%的成功受精和发育条件下，将非渗透性小分子加载到未受精的海胆卵悬液中。穿孔在模拟细胞内环境的低钙培养基中进行。在这种培养基中，诱导形成的孔会保持开放几分钟，从而使细胞得以加载；通过向培养基中重新添加毫摩尔浓度的钙离子实现重新封闭。当孔开放时，外源性分子会流入，内源性代谢物会流出，并且卵最多可损失其ATP含量的40%但仍能存活。引入的代谢物会被细胞利用，例如，引入的3H-胸腺嘧啶核苷会掺入DNA中。该程序对于将非渗透性底物加载到卵中、允许在体内评估代谢以及引入其他有趣的非渗透性分子（如抑制剂、荧光指示剂等）将是有用的。尽管细节可能有所不同，但在类似细胞内的培养基中进行电穿孔的原理可能被证明对于以最小的活力损失加载其他细胞类型是有用的。
