Development of a novel electroporation method for the oyster parasite Perkinsus marinus.
Development of a novel electroporation method for the oyster parasite Perkinsus marinus.
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DOI：
10.1038/s41598-022-24548-0
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发表时间：
2022-11-21
影响因子：
4.6
通讯作者：
Hikosaka, Kenji
中科院分区：
文献类型：
--
作者：
Sakamoto, Hirokazu;Lin, Xiaoxia X.;Bai, Yun D.;Chen, Xue F.;Zhang, Ziyue Z.;Honjo, Yui;Hikosaka, Kenji
基因操作技术是分子生物学的基础，并不断得到改进。然而，许多单细胞真核生物（原生生物）的基因转染方法尚未建立，从而阻碍了分子生物学研究。牡蛎寄生虫Perkinisus marinus是为数不多的建立基因转染和药物选择的原生生物之一。然而，目前的方案是繁琐的，需要特定的电穿孔器和脉冲条件，这限制了该技术在不同研究组中的可及性。在这里，我们提出了替代缓冲液和电穿孔的条件，使协议的限制较少。我们揭示了能够使用因杰尼奥电穿孔缓冲液和NEPA 21电穿孔仪将质粒导入海原藻细胞的脉冲条件。我们发现细胞数量和质粒浓度是电穿孔系统的关键参数。我们还构建了一个更简单的表达质粒，该质粒去除了寄生虫中用于基因表达的不必要区域。本研究结果解决了海原甲藻电穿孔的设备限制，为其他原生生物，特别是其他帕金索虫寄生虫和核心甲藻的电穿孔研究提供了参考。
Gene manipulation techniques are fundamental to molecular biology and are continuously being improved. However, gene transfection methods are not established for many unicellular eukaryotes (protists), thereby hindering molecular biological investigations. The oyster parasite Perkinisus marinus is one of the few protists with established gene transfection and drug selection. Nevertheless, the present protocols are tedious, requiring a specific electroporator and pulse conditions which limits the accessibility of this technique across different research groups. Here, we present alternative buffer and electroporation conditions that make the protocol less restrictive. We revealed the pulse condition that enables the introduction of plasmids into P. marinus cell using Ingenio electroporation buffer and NEPA21 electroporator. We found that number of cells and plasmid concentration were critical parameters for the electroporation system. We also constructed a simpler expression plasmid that is removed needless regions for gene expression in the parasite. Our findings resolved the equipment restriction in electroporation of P. marinus and would be a good reference for electroporation in other protists, in particular other Perkinsozoa parasites and core dinoflagellates.
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影响因子：
4.6
作者：
Carnegie RB;Ford SE;Crockett RK;Kingsley-Smith PR;Bienlien LM;Safi LSL;Whitefleet-Smith LA;Burreson EM
通讯作者：
Burreson EM
影响因子：
4
作者：
Fernández Robledo JA;Caler E;Matsuzaki M;Keeling PJ;Shanmugam D;Roos DS;Vasta GR
通讯作者：
Vasta GR
DOI：
10.1016/j.cbpb.2022.110758
发表时间：
2022-05-26
影响因子：
2.2
作者：
Fraga, Dean;Ellington, W. Ross;Suzuki, Tomohiko
通讯作者：
Suzuki, Tomohiko
影响因子：
2.5
作者：
Einarsson, Elin;Lassadi, Imen;Waller, Ross F.
通讯作者：
Waller, Ross F.
影响因子：
5.2
作者：
Reñé A;Alacid E;Gallisai R;Chambouvet A;Fernández-Valero AD;Garcés E
通讯作者：
Garcés E