Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566, Japan.
Department of Biotechnology and Life Science, Tokyo University Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
Sci Rep. 2019 Feb 15;9(1):2163. doi: 10.1038/s41598-018-38119-9.
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.
植物的基因组工程在很大程度上依赖于有效分子技术的可用性。尽管进行了大量的研究，但植物的基因组工程在基因传递方面仍然受到限制，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，我们描述了一种使用电穿孔介导的蛋白质递送到具有完整细胞壁的培养拟南芥细胞中的方法，并证明了 Cre 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，我们能够实现高效、低毒的蛋白质递送到拟南芥细胞中，尽管存在细胞壁，但效率仍达到 83%。据我们所知，这是第一个报道证明电穿孔介导的 Cre 重组酶蛋白质递送到具有完整细胞壁的植物细胞中实现无核酸基因组工程的报告。
