Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.

Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like fluorescence microscope, is usually required for the visualization of GFP, limitings its application to fixed locations in samples. A reporter that can be visualized in real-time regardless the shape, size and location of the target samples will increase the flexibility and efficiency of research work. Here, we report the application of a GFP-like protein, called eYGFPuv, in both transient expression and stable transformation, in two herbaceous plant species (Arabidopsis and tobacco) and two woody plant species (poplar and citrus). We observed bright fluorescence under UV light in all of the four plant species without any effects on plant growth or development. eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues. With a focus on in vitro transformation, we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identified visually during the callus stage and the shoot stage, enabling early and efficient selection of transformants. Furthermore, whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants. In addition, our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics. This work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3′-end phosphate. This activity has not been reported in plants. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2 depends on the signal peptide and ribonuclease catalytic residues, suggesting that secreted SRNs cleave RNAs in apoplast and are detected by the host. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

Bioactivity of Recombinant Humanized Monoclonal Antibody Against HER2 and Its Mechanism of Action on Ovarian Cancer

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) protein is overexpressed on the surface of various epithelial ovarian cancer tissues, which mediates the proliferation, differentiation, metastasis and signal transduction of tumor cells and is a potential cancer therapeutic target.METHODS: In this paper, the recombinant anti-HER2 humanized IgG1 monoclonal antibody (rhHer2-mAb) was expressed in HEK293F cells by constructing mammalian cell expression vector and optimizing transfection conditions. The antibody was purified by rProtein A affinity chromatography, and its mediated antibody dependent cytotoxicity (ADCC) was identified by lactate dehydrogenase(LDH) lactate dehydrogenase release assay. The anti-tumor activity of rhHer2-mAb was evaluated in NOD/SCID mice. RESULTS: The expression of rhHer2-mAb in HEK293F cells was at the highest level (100.5 mg/L) when the ratios of DNA/PEI and light chain/heavy chain was 1:4 and 1:2, respectively. The IC50 on ADCC of antibodies against SK-OV-3, OVCAR-3 and A-2780 cells were 12.36, 5.43 and 102.90ng/ml, respectively. Animal experiments in mice showed that rhHer2-mAb could effectively inhibit the growth of SK-OV-3 tumor at the dose of 10 mg/kg.CONCLUSIONS: The recombinant monoclonal antibody was obtained by transient gene expression technology and its bioactivity was studied in vitro and in vivo , providing a novel insight for the development and production of future biotechnology-based drugs using transient gene expression technology of HEK293F.

The Powdery Mildew Effector CSEP0027 Interacts With Barley Catalase to Regulate Host Immunity

Powdery mildew is one of the most important fungal pathogen diseases. The genome of barley mildew fungus, Blumeria graminis f. sp. hordei (Bgh), encodes a large number of candidate secreted effector proteins (CSEPs). So far, the function and mechanism of most CSEPs remain largely unknown. Here, we identify a Bgh effector CSEP0027, a member of family 41, triggering cell death in Nicotiana benthamiana. CSEP0027 contains a functional signal peptide (SP), verified by yeast secretion assay. We show that CSEP0027 promotes Bgh virulence in barley infection using transient gene expression and host-induced gene silencing (HIGS). Barley catalase HvCAT1 is identified as a CSEP0027 interactor by yeast two-hybrid (Y2H) screening, and the interaction is verified in yeast, in vitro and in vivo. The coexpression of CSEP0027 and HvCAT1 in barley cells results in altered localization of HvCAT1 from the peroxisome to the nucleus. Barley stripe mosaic virus (BSMV)-silencing and transiently-induced gene silencing (TIGS) assays reveal that HvCAT1 is required for barley immunity against Bgh. We propose that CSEP0027 interacts with barley HvCAT1 to regulate the host immunity and likely reactive oxygen species (ROS) homeostasis to promote fungal virulence during barley infection.