A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

Abstract

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.

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology

“…Batch electroporation (EP) is an alternative method to directly deliver proteins into cultured mammalian cells (Lambert et al, 1990; Chakrabarti et al, 1989; Vienken et al, 1978; Shi et al, 2018; Furuhata et al, 2019). EP-mediated protein transduction has surged in recent years with applications ranging from, cellular structural biology and the delivery of isotopically labeled proteins for in-cell NMR measurements (Theillet et al, 2016) to the introduction of immunoglobulins for targeting and degradation of endogenous proteins (Clift et al, 2017), and the delivery of ribonucleotide particles (RNPs) for CRISPR-Cas9-mediated gene editing (Kim et al, 2014; Lin et al, 2014; Liang et al, 2015; Hashimoto and Takemoto, 2015).…”

Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system

“…The GUS gene encodes β-glucuronidase that catalyzes the hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides. GUS-expressing cells and tissue can be visualized either by staining with a blue dye using X-Gluc or by the emitted fluorescence via utilization of the 6-chloro-4-methylumbelliferyl-β-D-glucuronide (CMUG) fluorogenic substrate [23,26]. Although these techniques enable the spatial visualization and quantification of gene expression, they are both invasive and time-consuming.…”

“…The A. thaliana T87 strain (RIKEN Bio Resource Center, Ibaraki, Japan) was maintained and transformed with Agrobacterium (Rhizobium radiobacter) according to a previously described protocol [23]. Briefly, the T87 cells were co-cultured with Agrobacterium cells carrying the binary plasmids for 2 d and selected by plating on hygromycin-containing agar.…”

Electroporation-mediated genetic transformation of oil palm (Elaeis guineensis)

“…Although plant and algae cells behave differently during their development, the reports showed that cell wall barriers can be penetrated as long as the electroporation conditions are optimized. In addition, Furuhata et al (2019) performed electroporation-mediated protein delivery into cultured Arabidopsis cells. The Cre recombinase was successfully transferred through the cell wall with high efficiency.…”