Chan Ching Man, Miller Andrew L, Webb Sarah E
Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
Cold Spring Harb Protoc. 2013 May 1;2013(5):456-60. doi: 10.1101/pdb.prot072975.
When holo-aequorin is injected into zebrafish embryos at the one-cell stage, it is normally depleted by ~24 h post-fertilization (hpf). In order to acquire Ca(2+) signaling information from embryos older than 24 hpf, we have developed a protocol to express apoaequorin transiently in embryos, after which we reconstitute active holo-aequorin in vivo by introducing the cofactor coelenterazine into the developing embryo. This protocol describes the preparation of apoaequorin mRNA, followed by microinjection into embryos and incubation with coelenterazine to reconstitute holo-aequorin.
当在单细胞期将全水母发光蛋白注射到斑马鱼胚胎中时，它通常在受精后约24小时（hpf）被耗尽。为了从大于24 hpf的胚胎中获取Ca(2+)信号信息，我们开发了一种在胚胎中瞬时表达脱辅基水母发光蛋白的方案，之后通过将辅因子腔肠素引入发育中的胚胎在体内重建活性全水母发光蛋白。本方案描述了脱辅基水母发光蛋白mRNA的制备，随后显微注射到胚胎中，并与腔肠素孵育以重建全水母发光蛋白。
