Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development
Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development
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DOI：
10.1387/ijdb.062151cc
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发表时间：
2006-01-01
影响因子：
0.7
通讯作者：
Miller, Andrew L.
中科院分区：
文献类型：
--
作者：
Cheung, Chris Y.;Webb, Sarah E.;Miller, Andrew L.
当水母发光蛋白被显微注射到卵裂期的斑马鱼胚胎中时，它在大约24小时内就被大量消耗。因此，目前不可能使用这种方法对斑马鱼发育后期的Ca 2+信号进行成像。因此，我们开发了表达脱辅基水母发光蛋白的方案，即，水母发光蛋白的蛋白质成分，在斑马鱼胚胎中瞬时表达，然后通过将腔肠素辅因子分别加载到胚胎中在体内重建完整的水母发光蛋白。两种类型的脱辅基水母发光蛋白mRNA，aeq-mRNA和aeq：：EGFP-mRNA，后者含有增强的绿色荧光蛋白（EGFP）序列，在体外转录，当这些被显微注射到胚胎中，他们成功地翻译脱辅基水母发光蛋白和脱辅基水母发光蛋白和EGFP的融合蛋白（脱辅基水母发光蛋白-EGFP），分别。我们发现，aeq：：EGFP mRNA比等量的aeq-mRNA对胚胎的毒性更大。此外，在体外重建试验中，脱辅基水母发光蛋白-EGFP产生的发光比脱辅基水母发光蛋白少，与腔肠素重建后，并与Ca 2+的添加。此外，当对表达脱辅基水母发光蛋白的完整腔肠素装载胚胎成像时，观察到从类似于2.5至48 hpf的Ca 2+信号，这些信号的时空模式高达24 hpf，与用水母发光蛋白观察到的相当。这种使用aeq-mRNA的瞬时水母发光蛋白表达方法为监测斑马鱼发育的24-48 hpf期间的Ca 2+信号提供了有价值的工具。因此，它有效地将基于水母发光蛋白的Ca 2+成像窗口延长了额外的24小时。
When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by similar to 24 hours. Thus, it is currently not possible to image Ca2+ signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca2+. Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca2+ signals from similar to 2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca2+ signaling during the 24-48 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca2+ imaging window by an additional 24 hours.