Manipulation of Gene Activity in the Regenerative Model Sea Anemone, Nematostella vectensis.
Manipulation of Gene Activity in the Regenerative Model Sea Anemone, Nematostella vectensis.
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再生模型海葵 Nematostella vectensis 中基因活性的操纵。
DOI：
10.1007/978-1-0716-2172-1_23
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Gibson,MatthewC
中科院分区：
文献类型：
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作者：
Hill,EricM;Chen,Cheng-Yi;DelViso,Florencia;Ellington,LaceyR;He,Shuonan;Karabulut,Ahmet;Paulson,Ariel;Gibson,MatthewC
凭借令人惊讶的复杂基因组和不断扩展的遗传工具包，海葵Nematostella vectensis已成为研究发育和全身再生的强大模型系统。在这里，我们提供了在该系统中短发夹RNA（shRNA）介导的基因敲减和CRISPR/Cas9靶向诱变的最新方案。我们进一步表明，一个简单的Klenow反应，然后在体外转录允许生产基因特异性的shRNA和单向导RNA（sgRNA）在一个快速，负担得起的，容易扩展的方式。总之，shRNA敲除和CRISPR/Cas9靶向诱变允许快速筛选基因功能以及产生稳定的突变株系，从而能够在整个Nematostella生命周期中进行功能遗传分析。
With a surprisingly complex genome and an ever-expanding genetic toolkit, the sea anemone Nematostella vectensis has become a powerful model system for the study of both development and whole-body regeneration. Here we provide the most current protocols for short-hairpin RNA (shRNA)-mediated gene knockdown and CRISPR/Cas9-targeted mutagenesis in this system. We further show that a simple Klenow reaction followed by in vitro transcription allows for the production of gene-specific shRNAs and single guide RNAs (sgRNAs) in a fast, affordable, and readily scalable manner. Together, shRNA knockdown and CRISPR/Cas9-targeted mutagenesis allow for rapid screens of gene function as well as the production of stable mutant lines that enable functional genetic analysis throughout the Nematostella life cycle.