Genome engineering in plants might be challenging due to thick cell wall and low efficiency of DNA or protein delivery into the cell. One of the approaches that allow the injection of biomaterial in plant cells is electroporation. Herein, plant cells get exposed to brief electrical pulses, forming microscopic perforations in cell wall and allowing biomaterial to enter the protoplast.
Furuhata et al (2019) developed a new system for delivery of proteins into plant cells. They optimized the electroporation conditions in Arabidopsis thaliana cells and achieved efficient as well as less-toxic protein delivery in the cells.
The model protein used in this study was the Cre recombinase, a popular genome engineering tool. This enzyme is part of the Cre/lox system, which allows removal of selectable markers from genome. In the study cited below, the host cell line was engineered in order to contain two lox sites at both ends of the gfp gene coding for a fluorescent reporter, followed by a coding sequence for ß-glucuronidase (GUS) with inactive promoter. When Cre recombinase is delivered to the cytoplasm, it excises the gfp gene at lox sites, leading to a construct containing GUS gene with functional promoter, allowing its expression. The cells transformed with Cre were screened for the GUS activity using chromogenic substrate X-glucuronide (X-gluc) from Carbosynth. X-gluc was dissolved in staining buffer containing methanol and sodium phosphate and incubated with cells at 37 ⁰C. The development of dark blue precipitates indicated the expression of GUS and therefore successful Cre delivery into intracellular space.
For more detail, read the original paper published in Nature Scientific Reports: Furuhata et al., 2019.