title: Reconstitution of holo-aequorin with apoaequorin mRNA and coelenterazine in zebrafish embryos.
abstract: 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.
authors: 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.
authors: Ching Man
authors: CM
authors: Chan
authors: Andrew L
authors: AL
authors: Miller
authors: Sarah E
authors: SE
authors: Webb
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D015870:Gene Expression; D007093:Imidazoles / Q000378:metabolism*; D008845:Microinjections / Q000379:methods; D011719:Pyrazines / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology*
chemical_list: D001059:Apoproteins; D007093:Imidazoles; D011719:Pyrazines; D012333:RNA, Messenger; D011994:Recombinant Proteins; C060014:apoaequorin; C017144:coelenterazine; D000331:Aequorin