Proteolistics: A Protein Delivery Method.

Intracellular protein delivery in plant tissues is becoming an important tool for addressing both basic and applied research questions by plant biologists, especially in the era of genome editing. The ability to deliver proteins or protein/RNA complexes into cells allows for producing gene-edited plants that are free of transgene integration in the genome. Here we describe a protocol for the delivery of a protein/gold particle mixture in plant cells through biolistics. The key for the delivery is the drying of the protein/gold suspension directly onto the gene-gun cartridge or macrocarrier. The intracellular protein delivery into plant cells is achieved through the bombardment using the Bio-Rad PDS-1000/He particle delivery device. We termed this methodology "proteolistics."

Proteolistics: a biolistic method for intracellular delivery of proteins.

In this work, an intracellular protein delivery methodology termed "proteolistics" is described. This method utilizes a biolistic gun apparatus and involves a simple protein/projectile preparation step. The protein to be delivered is mixed with a gold particle microprojectile suspension and is placed onto a gene gun cartridge, where it is dehydrated using either lyophilization or room-temperature air-drying. Subsequent intracellular protein delivery is achieved in plant and mammalian tissues upon bombardment. Because the method does not require modification of delivery agents or cargo biomolecules and involves a simple physical deposition of the protein onto the microprojectiles, there is no restriction on protein type in terms of molecular weight, isoelectric point or tertiary structure. Because the method delivers protein through the widely used gene gun system, it can be readily applied to any tissue or organism amenable to biolistics. A variety of proteins with molecular weight ranging from 24 to 68 kDa and isoelectric point from 4.8 to 10.1 were tested in this work. It is anticipated that this simple and versatile technique will offer biologists a powerful tool for basic research in areas such as understanding of cell and gene functions and for biotechnological applications such as genome editing.

Parameters affecting the efficient delivery of mesoporous silica nanoparticle materials and gold nanorods into plant tissues by the biolistic method.

Applying nanotechnology to plant science requires efficient systems for the delivery of nanoparticles (NPs) to plant cells and tissues. The presence of a cell wall in plant cells makes it challenging to extend the NP delivery methods available for animal research. In this work, research is presented which establishes an efficient NP delivery system for plant tissues using the biolistic method. It is shown that the biolistic delivery of mesoporous silica nanoparticle (MSN) materials can be improved by increasing the density of MSNs through gold plating. Additionally, a DNA-coating protocol is used based on calcium chloride and spermidine for MSN and gold nanorods to enhance the NP-mediated DNA delivery. Furthermore, the drastic improvement of NP delivery is demonstrated when the particles are combined with 0.6 µm gold particles during bombardment. The methodology described provides a system for the efficient delivery of NPs into plant cells using the biolistic method.