Gharbiah Maey, Cooley James, Leise Esther M, Nakamoto Ayaki, Rabinowitz Jeremy S, Lambert J David, Nagy Lisa M
Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
Cold Spring Harb Protoc. 2009 Apr;2009(4):pdb.prot5185. doi: 10.1101/pdb.prot5185.
The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Intracellular microinjection is an important tool, especially for lineage tracing and perturbations of specific genes by knockdown approaches and synthetic mRNA injections. Two methods for the introduction of lineage tracers into particular cells are routine in Ilyanassa. Iontophoresis of charged molecules, such as fluorophore-dextran conjugates can be accomplished using a simply built current generator. Injection of an oil-based solution containing the fluorescent probe 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) is also straightforward. However, injection of oil-based solutions and iontophoresis have not been useful for delivering water-soluble reagents to perturb gene function, and pressure injection of aqueous solutions has been more challenging. This protocol describes a recently optimized procedure for the pressure injection of aqueous solutions into Ilyanassa embryos and zygotes with high rates of survival and normal development. The key parameters seem to be the injection needles, injection media, and the stage of injected embryos.
海生腹足纲动物伊利亚那沙螺（Ilyanassa obsoleta）是胚胎发育研究中一个长期且非常有用的模型。它是螺旋动物发育研究以及不对称细胞分裂研究中尤为重要的模型。这些胚胎适用于经典的胚胎学操作技术，以及越来越多的分子方法。伊利亚那沙螺也是变态发育、寄生生态学、环境污染物对形态和性功能的影响以及比较神经生物学研究的重要模型。细胞内微注射是一种重要工具，特别是用于谱系追踪以及通过基因敲低方法和合成mRNA注射对特定基因进行干扰。在伊利亚那沙螺中，将谱系示踪剂引入特定细胞的两种方法是常规方法。使用简单构建的电流发生器就可以实现带电分子（如荧光团 - 葡聚糖缀合物）的离子电渗疗法。注射含有荧光探针1,1 - 二辛基 - 3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiI）的油基溶液也很简单。然而，油基溶液注射和离子电渗疗法对于递送水溶性试剂以干扰基因功能并无用处，而水溶液的压力注射则更具挑战性。本方案描述了一种最近优化的程序，用于将水溶液压力注射到伊利亚那沙螺胚胎和受精卵中，具有高存活率和正常发育率。关键参数似乎是注射针、注射介质以及注射胚胎的阶段。
