Protocol For Transient Expression Research Articles

Quantitative Estimation of Promoter Activity in Cannabis sativa Using Agroinfiltration-Based Transient Gene Expression.

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Production and Purification of Virus-Like Particles by Transient Expression in Plants.

Transient expression in plants has become a useful production system for virus-like particle (VLP) expression. High yields and flexible approaches to assembling complex VLPs, combine with ease of scale-up and inexpensive reagents to provide an attractive method for recombinant protein expression in general. Plants have demonstrated excellent capacity for the assembly and production of protein cages for use in vaccine design and nanotechnology. Furthermore, numerous virus structures have now been determined using plant-expressed VLPs, showing the utility of this approach in structural virology. Transient protein expression in plants uses common microbiology techniques, leading to a straightforward transformation procedure that does not result in stable transgenesis. In this chapter, we aim to provide a generic protocol for transient expression of VLPs in Nicotiana benthamiana using soil-free plant cultivation and a simple vacuum infiltration procedure, along with methodology for purifying VLPs from plant leaves.

Highly efficient transient gene expression of three tissues in Osmanthus fragrans mediated by Agrobacterium tumefaciens

As one of the 10 most famous flowers, sweet osmanthus (Osmanthus fragrans) is widely utilized in landscaping and food processing in China. Owing to the high complexity and large size of its genome, it is still an enormous challenge to develop transgenic technology for O. fragrans. Therefore, it is necessary to develop an efficient transient expression protocol for the rapid identification of gene functions in O. fragrans. In this study, we selected three different tissues of O. fragrans, including calli, floral buds, and leaves, as explants for transient transformation mediated by Agrobacterium tumefaciens. However, we found that one protocol did not work for all tissues. Calli were induced and proliferated from the immature plumule of O. fragrans on one-half Murashige and Skoog media that contained 0.5 mg·L−1 6-benzylaminopurine and 1 mg·L−1 naphthalene acetic acid. GUS (β-glucuronidase) was used as a reporter gene, where its level of transcription reflected the abundance of Agrobacterium-mediated T-DNA transferred into explants. To obtain the optimal transient expression protocol for each explant, we evaluated the effects of Agrobacterium load, acetosyringone concentration, co-culture duration, bud bract removal, and explant preculture on the transformation efficiency. The use of our transient expression protocols to overexpress a transcriptional repressor of OfXTH14, OfMYB4, in calli, floral buds, and leaves showed that the relative levels of expression of OfXTH14 decreased significantly in all three of our selected tissues. Since the transformed explants exhibited a transient high level of expression of the target gene, affecting the transcription of related genes as well as the synthesis of related metabolites, they can be widely applied to identify gene functions and study the molecular regulatory mechanisms of O. fragrans. Moreover, the critical steps to significantly increase the transformation efficiency varied from tissue to tissue, providing a reference for the development of transient expression protocols in other plant species.

Modified expi293 cell culture system using piggyBac transposon enables efficient production of human FVIII.

Human blood coagulation factor VIII (hFVIII) is used in hemostatic and prophylactic treatment of patients with hemophilia A. Biotechnological innovations have enabled purification of the culture medium of rodent or human cells harboring the hFVIII expression cassette. However, cell lines express hFVIII protein derived from an exogenous expression vector at a lower level than most other proteins. Here, we describe hFVIII production using piggyBac transposon and the human-derived expi293F cell line. Use of a drug selection protocol, rather than transient expression protocol, allowed cells harboring hFVIII expression cassettes to efficiently produce hFVIII. In heterogeneous drug-selected cells, the production level was maintained even after multiple passages. The specific activity of the produced hFVIII was comparable to that of the commercial product and hFVIII derived from baby hamster kidney cells. We also applied codon optimization to the hFVIII open reading frame sequences in the transgene, which increased production of full-length hFVIII, but decreased production of B-domain-deleted human FVIII (BDD-hFVIII). Low transcriptional abundance of the hF8 transgene was observed in cells harboring codon-optimized BDD-hFVIII expression cassettes, which might partially contribute to decreased hFVIII production. The mechanism underlying these distinct outcomes may offer clues to highly efficient hFVIII protein production.

Carbon-nanotube for Transient Expression in Rice Calli

Transient gene expression is an important technique in gene functional analysis, protein production and in plants. However, traditional transient expression methods using Agrobacterium are time-consuming with low efficiency. In this study, we demonstrated the use of a single-walled carbon nanotube (SWCNT) to deliver 35S:mCherry:pCXSN plasmid into rice calli. This transient expression protocol used a plastic medical syringe to create the physical pressure to help the delivery of plasmid DNA into plant cells. This protocol is relatively easy to perform and low cost. The transient expression was observed under fluorescence microscopy, and the mCherry fluorescence signal was quantified. The plasmid DNA was delivered into the rice cell using a 3:1 ratio (plasmid: carbon nanotube). The result showed that the mCherry signal of carbon nanotube + plasmid DNA treatment was the highest signal at 3 days post-transformation and decreased to a lower signal at 6 days post-transformation. Moreover, the quantitative analysis of mCherry mean intensity revealed that the signal intensity of carbon nanotube + plasmid DNA treatment was the highest level, and significantly higher than the control treatments at 3 days post-transformation. Plasmid DNA can be transported easily into plant calli using this carbon nanotube transient expression protocol.

A Robust Protocol for CRISPR-Cas9 Gene Editing in Human Suspension Cell Lines.

The implementation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems in mammalian cells has sparked an exciting new era in targeted gene editing. CRISPR-Cas9 technologies allow gene function to be interrogated by gene deletions, mutations, and truncations, and by epitope tagging and promoter activity modulation. Many robust protocols have been published to date on CRISPR-Cas9 techniques, however, most of these focus on adherent cell lines. Suspension cell lines, typically of hematolymphoid origin, such as Jurkat, Daudi, and TOLEDO, pose unique challenges to the setup of CRISPR experiments. Here, using B cell lymphoma cells as a primary model, we describe a comprehensive protocol for targeted gene manipulations using the CRISPR-Cas9 system in suspension cells. We also highlight necessary optimization steps to make this approach universal to other suspension cell lines. We first describe a detailed protocol for transient expression of the Cas9 nuclease and guide RNAs. We then suggest workflows for obtaining single-cell clones and for screening for successful homozygous knockout (KO) clones in suspension lines. This protocol aims to serve as a comprehensive resource to facilitate gene editing experiments for users starting CRISPR-Cas9 gene editing protocols on suspension cell lines or those looking to optimize their current workflows. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Transient CRISPR Cas9-gRNA delivery for gene knockout by NeonTM electroporation Support Protocol 1: Designing and preparing gene-specific gRNA Support Protocol 2: Preparation of conditioned medium and culture vessels for single-cell FACS Alternate Protocol 1: Transient CRISPR Cas9-gRNA delivery for gene knockout by Nepagene electroporator Basic Protocol 2: FACS and single-cell clone generation Alternate Protocol 2: Manual cell dilution to obtain single-cell clones Basic Protocol 3: Confirming indels status in single-cell clones by PCR on genomic DNA and Sanger sequencing.

Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol

The transient gene expression of protoplasts in plant is a considerable tool for gene functional research that has been widely used in gene analysis and functional characterization. Therefore, the objectives of this study were to develop a protocol for the isolation and purification of sugarcane protoplasts (Saccharum spp. hybrids), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 × 106 protoplasts g−1 FW) and a survival rate of > 80.0% was achieved through enzymatic hydrolysis at constant temperature of 28 °C, 60–70 rpm min−1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG 4000 for 25 min and the ScD27 protein was localized in the chloroplasts. The localization of ScD27 protein in sugarcane protoplast demonstrated that the newly developed protocol was functionally effective. This optimized sugarcane protoplast isolation, purification, and transient expression protocol lays a foundation for future molecular biology research in sugarcane.

A modified transient gene expression protocol for subcellular protein localization analysis in rice

Transient protein expression is a useful technique for investigating the protein localization and functional analysis of stress responses in rice plants. Currently, available methods for in planta transient expression analysis include Agrobacterium-mediated transformation, protoplast transformation using polyethylene glycol or electroporation, and biolistic bombardment expression which have several disadvantages and are not well suited for the rice. Therefore, development of a method for rapid and efficient analysis of protein expression, subcellular localization, pathogen effector screening, and protein–protein interaction in rice is required. We developed a protocol for in planta gene expression analysis in sliced rice sheath cells by modifying and optimizing the biolistic particle bombardment technique. By obtaining thin sections (~400 μm) of rice sheath cells, auto-fluorescence from chlorophyll was eliminated. This system was validated through the localization of marker genes specifically expressed in nuclei, plasma membranes, and tonoplast. In addition, high transformation efficiency of 30% was achieved. Therefore, this protocol provides a new and rapid method for transient gene expression assay in rice. Protein secretion was examined in rice sheath cells using predicted secretory proteins from rice blast fungus, indicating that this method is applicable to plant–microbe interaction studies. The transient expression protocol established here is well optimized for protein localization, secretion, and host–pathogen protein interaction studies in rice. A typical experiment can be completed in three days.

Efficient mesophyll protoplast isolation and development of a transient expression system for castor-oil plant (Ricinus communis L.)

IntroductionWe investigated the main factors affecting the efficacy of protoplast isolation, including leaf-obtaining period, cutting shapes of leaf material, enzyme concentration, enzymolysis time, and centrifugal speed.MethodsProtoplast isolation was optimal on the condition of 20 days of leaf materials, 2-mm filament of leaves, 1.6% RS and 0.8% R-10, 80 min of enzymolysis, and 700 rpm of centrifugation, resulting in the best yield (1.19 X 106 protoplasts/g FW) and vitality (80.34%) of mesophyll protoplasts. The transient expression vector pGFPl with green fluorescent protein was transfected into the obtained protoplasts from castor by polyethylene glycol-mediated method with a transformation efficiency of 12.37%.ResultsMoreover, the applicability of the system for studying the subcellular localization of Re FATA (an acyl-ACP thioesterase) was validated via the protoplast isolation and transient expression protocol in this study.DiscussionCollectively, the efficient mesophyll protoplast isolation and protoplast transient expression system facilitate to analyze the function of specific gene in castor (Ricinus communis L).

Transient Expression of AMPK Heterotrimer Complexes in Mammalian Cells.

Regulation of AMP-activated protein kinase (AMPK) signalling is complex and involves contributions from adenine nucleotides, co-/posttranslational modifications, and isoform composition of the AMPK heterotrimer. It is becoming apparent that AMPK activation/inhibition by synthetic drugs involves similar levels of complexity. Major advances in our understanding of these mechanisms have been gained from recombinant expression systems that provide sufficient quantities of highly purified material for structure/function studies. Here, we provide a detailed protocol for transient expression of affinity-tagged AMPK complexes in mammalian cells. We have found this system to be optimal as a source of enzyme possessing regulatory modifications found in vivo.

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz)

BackgroundCassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced.MethodsFully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization.ResultsIn this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system.ConclusionWe optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Rab21 attenuates EGF-mediated MAPK signaling through enhancing EGFR internalization and degradation

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is regulated by endocytosis where many Rab proteins play an important role in the determination of the receptor recycle or degradation. In an effort to better understand how EGF signaling is regulated, we examined the role of Rab21 in regulation of the degradation and signal transduction of the EGFR. Using a transient expression protocol in HEK293T and HeLa cells, we found that Rab21 enhanced the degradation of EGFR through accelerating its internalization in both EGF-independent and EGF-dependent manners. We further demonstrated that Rab21 interacted with EGFR by immunoprecipitation experiments. Interestingly, we observed that overexpression of Rab21 attenuated EGF-mediated mitogen-activated protein kinase (MAPK) signaling by inducing EGFR degradation. Taken together, these data suggest that Rab21 plays a negative role in the EGF-mediated MAPK signaling pathway.

In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC)

INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.

Transformation of lentil (Lens culinaris M.) cotyledonary nodes by vacuum infiltration ofAgrobacterium tumefaciens

Lentil cotyledonary nodes are some of the most regenerative tissues in legumes. Attempts to transform them by vacuum filtration have been limitedly successful. This report describes a rapid and convenient transient expression protocol based on vacuum infiltration ofAgrobacterium cells into lentil cotyledonary nodes. Vacuum-infiltrated tissues had significantly (P<.05) higher transient GUS expression than did noninfiltrated tissues. Under optimal conditions (infiltration at 200 mmHg for 20 min), 95% of theAgrobacterium-infiltrated explants exhibited an average of 16 blue foci. We believe this to be the first report of this technique for transient gene expression in lentil cotyledonary nodes.

The clathrin endocytic pathway in viral infection.

How important is the clathrin-dependent endocytic pathway for entry of viruses into host cells? While it is widely accepted that Semliki Forest virus (SFV), an enveloped virus, requires this pathway there are conflicting data concerning the closely related Sindbis virus, as well as varying results with picornaviruses such as human rhinovirus 14 (HRV 14) and poliovirus. We have examined the entry mode of SFV, Sindbis virus, HRV 14 and poliovirus using a method that identifies single infected cells. This assay takes advantage of the observation that the clathrin-dependent endocytic pathway is specifically and potently arrested by overexpression of dynamin mutants that prevent clathrin-coated pit budding. Using HeLa cells and conditions of low multiplicity of infection to favor use of the most avid pathway of cell entry, it was found that SFV, Sindbis virus and HRV 14 require an active clathrin-dependent endocytic pathway for successful infection. In marked contrast, infection of HeLa cells by poliovirus did not appear to require the clathrin pathway.

RGS7 attenuates signal transduction through the G(alpha q) family of heterotrimeric G proteins in mammalian cells.

The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase-activating proteins (GAPs) on the G(alpha i) and G(alpha q) subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on G(alpha i1), and G(alpha o) heterotrimeric proteins and that RGS7 acts to down-regulate G(alpha q)-mediated calcium mobilization in a whole-cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and G(alpha q) mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in G(alpha q)-mediated signaling in the CNS.