A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall
A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall
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DOI：
10.1038/s41598-018-38119-9
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发表时间：
2019-02-15
影响因子：
4.6
通讯作者：
Kato, Yoshio
中科院分区：
文献类型：
--
作者：
Furuhata, Yuichi;Sakai, Ayako;Kato, Yoshio
植物基因组工程高度依赖于有效的分子技术的可用性。尽管进行了大量的研究，但植物基因组工程在基因递送方面仍然受到限制，这需要使用感染性细菌或苛刻的条件，因为难以通过细胞壁将生物材料递送到植物细胞中。在这里，我们描述了一种方法，使用电穿孔介导的蛋白质传递到培养的拟南芥细胞具有完整的细胞壁，并证明铬介导的位点特异性重组。通过优化电脉冲、蛋白质浓度和电穿孔缓冲液的条件，我们能够以83%的效率将蛋白质递送到拟南芥细胞中，尽管有细胞壁。据我们所知，这是第一份报告证明了Cre重组酶的电穿孔介导的蛋白质递送，以实现在具有完整细胞壁的植物细胞中的无核酸基因组工程。
Genome engineering in plants is highly dependent on the availability of effective molecular techniques. Despite vast quantities of research, genome engineering in plants is still limited in terms of gene delivery, which requires the use of infectious bacteria or harsh conditions owing to the difficulty delivering biomaterial into plant cells through the cell wall. Here, we describe a method that uses electroporation-mediated protein delivery into cultured Arabidopsis thaliana cells possessing an intact cell wall, and demonstrate Cre-mediated site-specific recombination. By optimizing conditions for the electric pulse, protein concentration, and electroporation buffer, we were able to achieve efficient and less-toxic protein delivery into Arabidopsis thaliana cells with 83% efficiency despite the cell wall. To the best of our knowledge, this is the first report demonstrating the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing an intact cell wall.