Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Abstract

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work 11 , and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy. Video LinkThe video component of this article can be found at

Effects of morphology and size of nanoscale drug carriers on cellular uptake and internalization process: a review

“…66 More research has been done on non-viral techniques, such as electroporation, gene guns, and sonoporation as mRNA delivery methods. 67,68 Although technically possible, utilizing such methods to manipulate cells ex vivo using mRNA transfection is time-consuming, costly, and generally unsuitable for widespread application. 68 Biomaterials have shown remarkable promise for transporting different biomacromolecules, including DNA and siRNA, with signicant improvement over the non-viral methods listed above.…”

Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases

“…As discussed above, the immunogenicity has been overcome by chemical modifications; however, the instability of mRNA under physiological conditions requires additional action. Electroporation, 271 gene gun, 272 microinjection, 273 and sonoporation 274 have been investigated for mRNA delivery; however, these are restricted to ex vivo manipulation and ill suited for systemic delivery. Therefore, suitable mRNA carriers should exhibit the following functions: protection from RNase degradation, evasion of direct renal clearance, avoidance of nonspecific interaction, facilitation of mRNA stability, and sufficient mRNA loading and release.…”

Hybridization Networks of mRNA and Branched RNA Hybrids

“…[27,28] Complexation can lead to bundles of mRNAs in polyion complexes that facilitate delivery into cells. [29] More conventional transfection experiments with mRNA have been performed with cationic liposomes, [30] electroporation, [31] or a "gene gun", [32] but such approaches are not suitable for clinical applications. Instead, biomaterial-or nanoparticle-based strategies are being sought.…”