Transient Transfection Of Mammalian Cells Research Articles

Quantitative Analysis of Siglec Ligands by Flow Cytometry.

Siglecs (sialic acid-binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor-type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi-quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec-Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec-Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec-Fc fusion protein Support Protocol 1: Sialidase treatment of mammalian cells Support Protocol 2: Preparation of recombinant Siglec-Fc fusion protein via transient transfection of mammalian cells.

Single Genomic Loci Labeling and Manipulation Using SIMBA System.

The SIMBA (Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies) system is an innovative CRISPR-based imaging technique that leverages dCas9-SunTag and FRB-mCherry-HP1α, with scFv-FKBP acting as a bridge. This powerful system enables simultaneous visualization and manipulation of genomic loci. The dCas9-SunTag fusion protein allows for precise targeting of specific genomic sites, and the FRB-mCherry-HP1α fusion protein facilitates the condensation of chromatin at the targeted loci. The scFv-FKBP bridge protein links dCas9-SunTag and FRB-mCherry-HP1α, ensuring efficient and specific recruitment of HP1α to the desired genomic loci. This integrated approach allows us to visualize and manipulate genomic regions of interest, opening up new avenues for studying genome organization, gene expression regulation, and chromatin dynamics in living cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cloning of genetic constructs Basic Protocol 2: Transient transfection in mammalian cells and live-cell imaging Basic Protocol 3: Generation of SIMBA-expressing stable cell lines Basic Protocol 4: Manipulation of genomic loci using SIMBA.

A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently invivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293T cells in both culture formats. DMEM was the best transfection medium for adherent HEK 293T cells, so we determined the kinetics of linear polyethyleneimine (LPEI) polyplex formation with plasmid DNA (pDNA) and subsequent cellular uptake. Statistical experimental designs revealed optimal transfection efficiency using 0.7 pg pDNA and 4.5 pg LPEI per cell. We used the amount of pDNA and LPEI per cell as the transfer criterion for HEK 293T/17 SF cell suspension cultures in FreeStyle 293 medium and confirmed optimal transfection at 1.1 pg pDNA and 6.6 pg LPEI per cell. We observed a strong correlation between polyplex size, transfection efficiency and post-transfection cell viability. Suspension cell transfection could be scaled to a 100-mL working volume without loss of efficiency. We conclude that pg pDNA and pg LPEI per cell is a suitable transfer criterion allowing the optimization of transient transfection using statistical experimental designs, thus minimizing the amount of pDNA and LPEI used without sacrificing transfection efficiency.

High-Throughput Screening Identifies Two Novel Small Molecule Enhancers of Recombinant Protein Expression.

As a primary strategy for production of biological drugs, recombinant proteins produced by transient transfection of mammalian cells are essential for both basic research and industrial production. Here, we established a high-throughput screening platform for improving the expression levels of recombinant proteins. In total, 10,011 small molecule compounds were screened through our platform. After two rounds of screening, we identified two compounds, Apicidin and M-344, that significantly enhanced recombinant protein expression. Both of the selected compounds were histone deacetylase inhibitors, suggesting that the two small molecules increased the expression levels of recombinant proteins by promoting histone acetylation. Moreover, both molecules showed low cytotoxicity. Therefore, our findings suggest that these small molecules may have wide applications in the future.

Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end-uses require significant amounts of material. In these cases, a single homogenous culture is usually more convenient, in terms of both consistency of expression and labour/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture volumes as high as 500L may be achieved in a single vessel. Here we describe the transfection of 293-6E cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.

High Throughput Transfection of HEK293 Cells for Transient Protein Production.

Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.

Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1×106 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96h until the product is harvested.

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

Optimization and automation of an end-to-end high throughput microscale transient protein production process.

High throughput protein production from transient transfection of mammalian cells is used in multiple facets of research and development studies. Commonly used formats for these high number expressions are 12-, 24- and 96-well plates at various volumes. However there are no published examples of a 96-deep well plate microscale (1,000 μL) suspension process for mammalian transient expression. For this reason, we aimed to determine the optimal operating conditions for a high producing, microscale HEK293 transient system. We evaluated the hydrodynamic flow and measured the oxygen transfer rate (OTR) and transient protein expression for 96-deep well plates of different well geometries filled at 600-1,000 μL working volumes and agitated at various speeds and orbital diameters. Ultimately, a round well-round bottom (RR) 96-deep well plate with a working volume of 1,000 µL agitated at 1,000 RPM and a 3 mm orbital diameter yielded the highest and most consistent total transient protein production. As plate cultures are subject to evaporation, water loss from different plate seals was measured to identify an optimal plate sealing method. Finally, to enable higher capacity protein production, both expression and purification processes were automated. Functionality of this end-to-end automation workflow was demonstrated with the generation of high levels of human IgG1 antibodies (≥360 µg/mL) with reproducible productivity, product quality and ≥78% purification recovery.

Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing.

Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.

Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection

Transient transfection of mammalian cells with plasmid expression vectors and chemical transfection reagents is widely used to study protein transport and dynamics as well as phenotypic alterations mediated by the overexpressed protein. Despite the undisputed impact of this technique, surprisingly little is known about the cellular effects mediated by the transfection process per se. Conceivably, off-target effects could have implications upon proteins or processes being studied and understanding the molecular pathways affected would add value to the interpretation of experimental observations subsequent to cell transfection.Here we have used a SILAC-based proteomic approach to study differentially expressed proteins after transfection of HeLa cells with ECFP vector using a commonly employed non-liposome based transfection reagent, Fugene®HD. Whereas the transfection reagent itself mediated minimal effects upon protein expression, 11 proteins were found to be significantly upregulated after transfection, all of which were associated with an interferon type I/II response. The upregulated proteins might potentially inflict major cellular processes such as RNA splicing, chromatin remodeling, post-translational protein modification and cell cycle control. The results were validated by western analysis as well as quantitative RT-PCR and this demonstrated that an essentially identical response was induced in HeLa by transfection using an empty pUC18 vector, which does not contain a mammalian virus promoter, as well as a liposome-based transfection reagent, LipofectamineTM2000. Notably, no induction of the interferon response was observed in HEK293 cells, suggesting that these cells might be preferable to HeLa to avoid undesired off-target effects in transfection studies encompassing interferon-signaling and antiviral responses.

Towards the development of a surface plasmon resonance assay to evaluate the glycosylation pattern of monoclonal antibodies using the extracellular domains of CD16a and CD64

We here report the production and purification of the extracellular domains of two Fcγ receptors, namely CD16a and CD64, by transient transfection in mammalian cells. The use of these two receptor ectodomains for the development of quantitative assays aiming at controlling the quality of monoclonal antibody production lots is then discussed. More specifically, the development of surface plasmon resonance-based biosensor assays for the evaluation of the glycosylation pattern and the aggregation state of monoclonal antibodies is presented. Our biosensor approach allows discriminating between antibodies harboring different galactosylation profiles as well as to detect low levels (i.e., less than 2%) of monoclonal antibody aggregates.

Development of a semi-automated high throughput transient transfection system

Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FXP liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

INVect - a novel polycationic reagent for transient transfection of mammalian cells

Background For rapid recombinant protein production in small to medium size volumes, transient transfection of mammalian cells is still the method of choice in biotechnology [1]. However, due to the high costs of commercially available lipofectamines or polycationic transfection reagents such as polyethylenimine (PEI), the most widely used transfection reagents available present a substantial economic bottleneck. While these reagents produce seemingly high transient transfection rates [2], there is still a strong desire for transfection reagents providing both secure and easy handling and higher recombinant protein production. As part of our commitment to excellence, InVivo BioTech Services initiated a joint venture with emp Biotech and developed a novel polycationic reagent, named INVect, for transient transfection and recombinant protein production in mammalian cells.

In vivo electroporation enhances immunogenicity and protection against influenza A virus challenge of an M2e-HSP70c DNA vaccine

There is a growing concern regarding continuous risk of emerging a new influenza pandemic. It is highlighted the need for novel vaccination techniques that quickly and effectively employed to respond to such threats. Although, DNA vaccine is a simple and effective approach to induce antigen specific immune responses, their potency requires further improvement. DNA vaccine encoding conserved antigen of influenza virus could provide protection in various animal models. Therefore, we constructed a plasmid vector encoding M2e-HSP70c sequences, pcDNA/MHc, as a candidate for universal influenza vaccine. The expression of newly constructed vectors was verified by transient transfection of mammalian cells (HEK293T cell line) and western blot analysis using commercial antibodies. Mice were injected subcutaneously (s.c.) by the help of electroporation (IEP) in the footpad area and boosted without IEP with 100μg of constructed vector. Furthermore, the potency of this construct to provoke humoral immune responses and its protectivity against lethal dose of viral challenge were evaluated. Based on our study, the fusion construct was immunogenic in mice and was able to confer both protection against lethal challenge of H1N1 virus and reduce viral load in lung homogenates of the infected mice.

Transduction of striatum and cortex tissues by adeno-associated viral vectors produced by herpes simplex virus- and baculovirus-based methods

Recombinant adeno-associated virus (AAV) vectors can be engineered to carry genetic material encoding therapeutic gene products that have demonstrated significant clinical promise. These viral vectors are typically produced in mammalian cells by the transient transfection of two or three plasmids encoding the AAV rep and cap genes, the adenovirus helper gene, and a gene of interest. Although this method can produce high-quality AAV vectors when used with multiple purification protocols, one critical limitation is the difficulty in scaling-up manufacturing, which poses a significant hurdle to the broad clinical utilization of AAV vectors. To address this challenge, recombinant herpes simplex virus type I (rHSV-1)- and recombinant baculovirus (rBac)-based methods have been established recently. These methods are more amenable to large-scale production of AAV vectors than methods using the transient transfection of mammalian cells. To investigate potential applications of AAV vectors produced by rHSV-1- or rBac-based platforms, the in vivo transduction of rHSV-1- or rBac-produced AAV serotype 2 (AAV2) vectors within the rat brain were examined by comparing them with vectors generated by the conventional transfection method. Injection of rHSV-1- or rBac-produced AAV vectors into rat striatum and cortex tissues revealed no differences in cellular tropism (i.e., predominantly neuronal targeting) or anteroposterior spread compared with AAV2 vectors produced by transient transfection. This report represents a step towards validating AAV vectors produced by the rHSV-1- and the rBac-based systems as promising tools, especially for delivering therapeutic molecules to the central nervous system.

Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure

Transient transfection of mammalian cells in suspension culture has recently emerged as a very useful method for production of research-scale quantities of recombinant proteins. The most commonly used cell lines for this purpose are suspension-adapted HEK and CHO cells. We report here that the plasma exposure in mice of an IL-23R extracellular domain Fc fusion protein (IL23R–Fc) differed dramatically depending on whether the protein was prepared by transient transfection of HEK or CHO cells. Specifically, IL23R–Fc expressed using CHO cells had about 30-fold higher in vivo plasma exposure compared to the HEK-expressed protein. In contrast to their differing plasma exposures, the HEK- and CHO-expressed proteins had equivalent in vitro biological activity. Characterization of the CHO- and HEK-expressed IL23R–Fc proteins indicated that the differences in in vivo plasma exposure between them are due to differential glycosylation.

The creation of stable cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 and generating Ebola virus-like particles utilizing an ecdysone inducible mammalian expression system

Ebolavirus is a filovirus that causes hemorrhagic fever in humans and is associated with case fatality rates of up to 90%. The lack of therapeutic interventions in combination with the threat of weaponizing this organism has enhanced research investigations. The expression of key viral proteins and the production of virus-like particles in mammalian systems are often pursued for characterization and functional studies. Common practice is to express these proteins through transient transfection of mammalian cells. Unfortunately the transfection reagents are expensive and the process is time consuming and labour intensive. This work describes utilizing an ecdysone inducible mammalian expression system to create stable cell lines that express the Ebolavirus transmembrane glycoprotein (GP), the soluble glycoprotein (sGP) and the matrix protein (VP40) individually as well as GP and VP40 simultaneously (for the production of virus like particles). These products were the same as those expressed by the transient system, by Western blot analysis and electron microscopy. The inducible system proved to be an improvement of the current technology by enhancing the cost effectiveness and simplifying the process.

At‐line monitoring of bioreactor protein production by surface plasmon resonance

An innovative and automated method for the at-line monitoring of secreted protein was developed by harnessing a Surface Plasmon Resonance-based biosensor to a bioreactor. The proof of concept was performed by following at-line the relative concentration of a secreted protein produced by transient transfection of mammalian cells in a bioreactor. Our results suggest that our approach can be readily applied to the at-line determination of both protein concentration and bioactivity. Our experimental setup and strategy can thus satisfy the needs related to the development of novel bioprocess control protocols in the context of the new process analytical technology that arises in the biopharmaceutical industry.