Development of the Myzozoan Aquatic Parasite Perkinsus marinus as A Versatile Experimental Genetic Model Organism
Development of the Myzozoan Aquatic Parasite Perkinsus marinus as A Versatile Experimental Genetic Model Organism
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DOI：
10.1016/j.protis.2021.125830
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发表时间：
2021-09-20
期刊：
影响因子：
2.5
通讯作者：
Waller, Ross F.
中科院分区：
文献类型：
--
作者：
Einarsson, Elin;Lassadi, Imen;Waller, Ross F.
Perkinsozoa 门是一种水生寄生虫谱系，对商业和自然软体动物种群具有毁灭性影响，还包括藻类、鱼类和两栖动物的寄生虫。它们与甲藻和顶端复门动物相关，因此为寄生虫学和进化研究提供了极好的遗传模型。先前已针对 Perkinsus spp 实现了遗传转化。但转基因表达工具很少，选择效果有限。我们试图使用 P. marinus 作为模型来扩展 Perkinsus 实验遗传工具的能力。我们构建了一个模块化质粒组装系统，用于同时表达多个基因。我们为嘌呤霉素、博来霉素和杀稻瘟素这三种药物开发了高效的选择系统，仅需三周即可发挥作用。我们开发了十一个具有可变表达强度的新启动子。此外，我们发现转基因的基因组整合主要是通过非同源重组，但也存在转基因片段化，包括某些元件的删除。为了应对这些动态过程，我们证明使用病毒 2A 肽的双顺反子转录本可以将选择与目标转基因表达的维持结合起来。总的来说，这些新工具和见解为基因改造和研究帕金苏斯作为水生寄生虫和进化模型提供了巨大的新能力。 (c) 2021 作者。由爱思唯尔有限公司出版。这是一篇基于 CC BY 许可证 (http://creativecommons.org/licenses/by/4.0/) 的开放获取文章。
The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).