Weisblat David A, Kuo Dian-Han
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
Cold Spring Harb Protoc. 2009 Apr;2009(4):pdb.prot5190. doi: 10.1101/pdb.prot5190.
One advantage of using Helobdella (leech) embryos as an experimental system is their amenability for microinjection. Blastomeres ranging in size from the zygote (400 microm diameter) down to micromeres and primary blast cells (approximately 20 microm diameter) can be injected by pressure under a dissecting microscope. Smaller cells can be injected by iontophoresis under a compound microscope. Microinjection is useful for studying embryonic development. For example, developmental fates of a cell can be followed by injecting a lineage tracer. A specific cell can be killed by injecting a toxic substance. Furthermore, cells can be killed at a given developmental stage by directing intense fluorescence illumination or a blue laser beam on fluorescein-labeled cells. Finally, gene expression can be manipulated in leech embryos by injecting zygotes or selected blastomeres with synthetic mRNA, morpholino antisense oligo, or a plasmid construct. Molecules <1500 Da can diffuse freely among early blastomeres via gap junctions. When intercellular diffusion of an injected substance is undesirable, small molecules should be conjugated to larger molecules such as dextran (10 kDa). Different commercial microinjection setups can be adopted for Helobdella embryos. This article describes how to microinject embryos using a versatile homemade pressure injection system. Under a dissecting microscope, embryos are immobilized by suction in a custom-fabricated chamber with the target cell facing upward. Cells are visualized using transillumination via a long-working-distance, dark-field condenser. The tip of a micropipette is brought into the target cell with a micromanipulator, and the injectant is delivered into the cell by pressure.
将扁蛭（水蛭）胚胎用作实验系统的一个优点是它们易于进行显微注射。从合子（直径400微米）到小分裂球和初级胚细胞（直径约20微米）大小不等的卵裂球都可以在解剖显微镜下通过压力进行注射。较小的细胞可以在复式显微镜下通过离子电渗法进行注射。显微注射对于研究胚胎发育很有用。例如，通过注射谱系示踪剂可以追踪细胞的发育命运。通过注射有毒物质可以杀死特定的细胞。此外，通过将强荧光照明或蓝色激光束对准荧光素标记的细胞，可以在给定的发育阶段杀死细胞。最后，通过向合子或选定的卵裂球注射合成mRNA、吗啉代反义寡核苷酸或质粒构建体，可以操纵水蛭胚胎中的基因表达。分子量小于1500道尔顿的分子可以通过间隙连接在早期卵裂球之间自由扩散。当不希望注射物质发生细胞间扩散时，小分子应与较大的分子如葡聚糖（10 kDa）偶联。对于扁蛭胚胎，可以采用不同的商业显微注射装置。本文描述了如何使用一种通用的自制压力注射系统对胚胎进行显微注射。在解剖显微镜下，胚胎通过吸力固定在定制的腔室中，目标细胞朝上。通过长工作距离暗场聚光器利用透照法观察细胞。用显微操作器将微量移液器的尖端移至目标细胞，然后通过压力将注射剂注入细胞。
