Kline Douglas
Department of Biological Sciences, Kent State University, Kent, OH, USA.
Methods Mol Biol. 2009;518:135-56. doi: 10.1007/978-1-59745-202-1_11.
Quantitative microinjection is used to introduce known quantities of molecules or probes into single cells to examine cellular function. The relatively large mammalian oocyte or egg is easily manipulated and can be injected with impermeant reagents including a variety of signaling molecules and fluorescent probes. Techniques have been developed to inject picoliter quantities of solution into oocytes and eggs with precision and reliability. The methods described here outline the quantitative injection procedures as they are used to inject mouse oocytes and eggs in a culture dish on the stage on an inverted microscope. The techniques are applicable to the oocytes, eggs, and early embryos of most mammalian species. Included are some general instructions on fabrication of transfer pipettes, holding pipettes, beveled injection pipettes, and equipment for quantitative injection.
定量显微注射用于将已知量的分子或探针导入单细胞以检查细胞功能。相对较大的哺乳动物卵母细胞或卵子易于操作，并且可以注射包括各种信号分子和荧光探针在内的非渗透性试剂。已经开发出将皮升量的溶液精确且可靠地注射到卵母细胞和卵子中的技术。这里描述的方法概述了用于在倒置显微镜载物台上的培养皿中注射小鼠卵母细胞和卵子的定量注射程序。这些技术适用于大多数哺乳动物物种的卵母细胞、卵子和早期胚胎。其中包括一些关于转移移液器、固定移液器、斜口注射移液器的制作以及定量注射设备的一般说明。
