Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis.
Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis.
复制标题
DOI：
10.1038/nprot.2013.009
复制
发表时间：
2013-05
期刊：
影响因子：
14.8
通讯作者：
Martindale MQ
中科院分区：
文献类型：
--
作者：
Layden MJ;Röttinger E;Wolenski FS;Gilmore TD;Martindale MQ
我们描述了一种胚胎显微注射的方案，用于新兴的模型系统，刺胞海葵，Nematostella vectensis。此外，我们还提供了通过显微注射体外翻译mrna或基因特异性寡核苷酸（Mos）来进行基因功能过表达和敲低的方案。我们的方法很简单，它利用了早期胚胎的自然粘附特性，将它们放置在聚苯乙烯培养皿上的单层中。胚胎在配备有荧光的解剖显微镜上观察，并使用picospritzer强制空气注射系统注射微注射针。显微操作器用于引导针头刺穿单个胚胎。注射时间约为1.5小时，经验丰富的研究人员可以一次注射约2000个胚胎。随着Nematostella基因组的公开，包括mrna的克隆和转录在内的整个方案可以在1周内完成。
We describe a protocol for microinjection of embryos for an emerging model system, the cnidarian sea anemone, Nematostella vectensis. In addition, we provide protocols for carrying out overexpression and knockdown of gene function through microinjection of in vitro–translated mRNAs or gene-specific oligonucleotide morpholinos (Mos), respectively. our approach is simple, and it takes advantage of the natural adherence properties of the early embryo to position them in a single layer on a polystyrene dish. embryos are visualized on a dissecting microscope equipped with epifluorescence and injected with microinjection needles using a picospritzer forced-air injection system. a micromanipulator is used to guide the needle to impale individual embryos. Injection takes ∼1.5 h, and an experienced researcher can inject ∼2,000 embryos in a single session. With the availability of the published Nematostella genome, the entire protocol, including cloning and transcription of mRNAs, can be carried out in ∼1 week.