Efficient Transient Expression of Plasmid DNA Using Poly (2-(N,N-Dimethylamino) Ethyl Methacrylate) in Plant Cells

Abstract

Nanomaterials have been widely studied for their potential to become the new generation of nanocarriers in gene transfection, yet it remains still difficult to apply them efficiently and succinctly to plant cells. Poly (2-(N,N-dimethylamino) ethyl methacrylate) (PDMAEMA), which possesses temperature and pH dual-sensitivity, has largely been applied in animal cells, but it is rarely involved in plant cells. As a proof of concept, PDMAEMA as a gene carrier is incubated with plasmid GFP (pGFP) to explore its transfection ability in plants, and cationic polymer polyethylenimine (PEI) is used as a control. pGFP was efficiently condensed into the nanostructure by electrostatic interactions at an N/P (amino group from cationic polymers/phosphate group from plasmid DNA (pDNA)) ratio of 15; after complexation into nanocarriers, pGFP was protected from endonuclease degradation according to the DNase I digestion assay. After incubation with protoplasts and leaves, GFP was observed with confocal microscopy in plant cells. Western blot experiments confirmed GFP expression at the protein level. Toxicity assay showed PDMAEMA had a lower toxicity than PEI. These results showed that transient expression of pGFP was readily achieved in Arabidopsis thaliana and Nicotiana benthamiana. Notably, PDMAEMA showed lower cytotoxicity than PEI upon incubation with Nicotiana benthamiana leaves. PDMAEMA exhibited great potency for DNA delivery in plant cells. This work provides us with new ideas of more concise and more effective methods for plant transformation.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts

“…Statistical significance analyses showed that incubation times between 15 and 60 min can be used with comparable efficiency. This finding differs from the Arabidopsis protoplast infection results, when a significantly longer incubation time was applied [21].…”

“…Statistical significance analyses showed that incubation times between 15 and 60 min can be used with comparable efficiency. This finding differs from the Arabidopsis protoplast infection results, when a significantly longer incubation time was applied [21]. As shown in Figure 4A,B, the incubation of maize protoplasts with polyplexes composed of plasmid DNA (pGFP, [25]) vector containing the GFP gene and the cationic polymer generated a considerable number of green fluorescent cells.…”

“…Since positively charged cationic polymers can interact with negatively charged DNA via electrostatic interactions, poly (2-(N,N-dimethylamino) ethyl methacrylate) (pDMAEM A) and polyethylenimine (PEI) have been widely used for DNA delivery in animal cells (see review by Bono et al [23]). Based on its lower toxicity, pDMAEMA has considerable potential for DNA delivery into plant cells [20,21]. In the present work, we have tested an additional cationic polymer, poly (2-hydroxypropylene imine) (PHPI), as a new alternative for nucleic acid delivery into plant protoplasts.…”

“…Regarding the application of cationic polymer-nucleic acid nanoparticle complexes, (polyplexes) in plant transformation, Finiuk et al [20] reported that pDMAEMA-based cationic polymers could transport plasmid DNA harboring the EYFP yellow fluorescent protein gene sequence into tobacco protoplasts. An et al [21] used condensed nanostructures obtained from the combination of plasmid GFP and cationic polymers, such as pDMAEMA or PEI, for incubation and consequent plasmid introduction into Arabidopsis thaliana and Nicotiana benthamiana protoplasts or leaves. After plasmid uptake, the transient expression of the marker gene was demonstrated through monitoring the GFP fluorescence.…”

Expression of PEI-coated gold nanoparticles carrying exogenous gene in periwinkle mesophyll cells and its practice in huanglongbing research

“…Clarity Western Substrate (BIO-RAD) was used for chemiluminescent detection of the GFP protein. The detection was performed using a LI-COR Odyssey Infrared Imaging System (Tanon, 5,200, China) ( An et al., 2022 ).…”