Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Preparation of Chitosan Nanoparticles through a Readily Solvent-Exchange Process for Efficient and Enhanced Gene Delivery.

In view of the excellent prospects of gene therapy and the potential safety and immunogenicity issues challenged by viral vectors, it is of great significance to develop a nonviral vector with low toxicity and low cost. In this work, we report a chitosan nanoparticle (CSNP) to be used as a gene vector prepared through a facile solvent-exchange strategy. Chitosan is first dissolved in ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIM Ac), and then, the solvent is exchanged with water/phosphate-buffered saline (PBS) to remove ionic liquid, forming a final CSNP dispersion after ultrasonication. The prepared CSNP shows a positive surface charge and can condense green fluorescent protein-encoding plasmid (pGFP) at weight ratios (CSNP/pGFP) of 5/1 or higher. Dynamic light scattering size and ζ-potential characterization and gel retardation results confirm the formation of CSNP/pGFP complexes. Compared with plain pGFP, efficient cellular internalization and significantly enhanced green fluorescent protein (GFP) expression are observed by using CSNP as a plasmid vector. Benefitting from the intrinsic biocompatibility, low cost, low immunogenicity, and abundant sources of chitosan, as well as the facile preparation and the efficient gene transfection capacity of CSNP, it is believed that this CSNP could be used as a nonviral gene vector with great clinical translational potentials.

Cationic Lipid Pairs Enhance Liver-to-Lung Tropism of Lipid Nanoparticles for In Vivo mRNA Delivery.

Much of current clinical interest has focused on mRNA therapeutics for the treatment of lung-associated diseases, such as infections, genetic disorders, and cancers. However, the safe and efficient delivery of mRNA therapeutics to the lungs, especially to different pulmonary cell types, is still a formidable challenge. In this paper, we proposed a cationic lipid pair (CLP) strategy, which utilized the liver-targeted ionizable lipid and its derived quaternary ammonium lipid as the CLP to improve liver-to-lung tropism of four-component lipid nanoparticles (LNPs) for in vivo mRNA delivery. Interestingly, the structure-activity investigation identified that using liver-targeted ionizable lipids with higher mRNA delivery performance and their derived lipid counterparts is the optimal CLP design for improving lung-targeted mRNA delivery. The CLP strategy was also verified to be universal and suitable for clinically available ionizable lipids such as SM-102 and ALC-0315 to develop lung-targeted LNP delivery systems. Moreover, we demonstrated that CLP-based LNPs were safe and exhibited potent mRNA transfection in pulmonary endothelial and epithelial cells. As a result, we provided a powerful CLP strategy for shifting the mRNA delivery preference of LNPs from the liver to the lungs, exhibiting great potential for broadening the application scenario of mRNA-based therapy.

An antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo.

The membrane-fusion-based internalization without lysosomal entrapment is advantageous for intracellular delivery over endocytosis. However, protein corona formed on the membrane-fusogenic liposome surface converts its membrane-fusion performance to lysosome-dependent endocytosis, causing poorer delivery efficiency in biological conditions. Herein, we develop an antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo. Leveraging specific lipid composition at an optimized ratio, such antifouling membrane-fusogenic liposome facilitates fusion capacity even in protein-rich conditions, attributed to the copious zwitterionic phosphorylcholine groups for protein-adsorption resistance. Consequently, the antifouling membrane-fusogenic liposome demonstrates robust membrane-fusion-mediated delivery in the medium with up to 38% fetal bovine serum, outclassing two traditional membrane-fusogenic liposomes effective at 4% and 6% concentrations. When injected into mice, antifouling membrane-fusogenic liposomes can keep their membrane-fusion-transportation behaviors, thereby achieving efficient luciferase transfection and enhancing gene-editing-mediated viral inhibition. This study provides a promising tool for effective intracellular delivery under complex physiological environments, enlightening future nanomedicine design.

Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

Possibilities and efficiency of MSC co-transfection for gene therapy.

Mesenchymal stem/stromal cells (MSCs) are not only capable of self-renewal, trans-differentiation, homing to damaged tissue sites and immunomodulation by secretion of trophic factors but are also easy to isolate and expand. Because of these characteristics, they are used in numerous clinical trials for cell therapy including immune and neurological disorders, diabetes, bone and cartilage diseases and myocardial infarction. However, not all trials have successful outcomes, due to unfavourable microenvironmental factors and the heterogenous nature of MSCs. Therefore, genetic manipulation of MSCs can increase their prospect. Currently, most studies focus on single transfection with one gene. Even though the introduction of more than one gene increases the complexity, it also increases the effectivity as different mechanism are triggered, leading to a synergistic effect. In this review we focus on the methodology and efficiency of co-transfection, as well as the opportunities and pitfalls of these genetically engineered cells for therapy.

Dermaseptin B2 bioactive gene's potential for anticancer and anti-proliferative effect is linked to the regulation of the BAX/BBC3/AKT pathway.

Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.

Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.

BACKGROUND: Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines. RESULTS: We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved. CONCLUSIONS: The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.

Transfected SARS-CoV-2 spike DNA for mammalian cell expression inhibits p53 activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2 proteins in cancer cells and increases cancer cell viability after chemotherapy exposure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike were found to have increased cell viability as compared to control cells. Further observations on γ-H2AX expression in spike-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway. The preliminary observations reported here warrant further studies to unravel the impact of SARS-CoV-2 and its various encoded proteins including spike on pathways of tumorigenesis and response to cancer therapeutics. More efforts should be directed at studying the effects of the SARS-CoV-2 spike and other viral proteins on host DNA damage sensing, response and repair mechanisms. A goal would be to understand the structural basis for maximal anti-viral immunity while minimizing suppression of host defenses including the p53 DNA damage response and tumor suppression pathway. Such directions are relevant and important including not only in the context of viral infection and mRNA vaccines in general but also for patients with cancer who may be receiving cytotoxic or other cancer treatments.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Recombinant human fibroblast growth factor 7 obtained from stable Chinese hamster ovary cells enhances wound healing.

Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7.

Intracellular Delivery of Plasmid DNA Using Amphipathic Helical Cell-Penetrating Peptides Containing Dipropylglycine.

Cell-penetrating peptides (CPPs) serve as potent vehicles for delivering membrane-impermeable compounds, including nucleic acids, into cells. In a previous study, we reported the successful intracellular delivery of small interfering RNAs (siRNAs) with negligible cytotoxicity using a peptide containing an unnatural amino acid (dipropylglycine). In the present study, we employed the same seven peptides as the previous study to evaluate their efficacy in delivering plasmid DNA (pDNA) intracellularly. Although pDNA and siRNA are nucleic acids, they differ in size and biological function, which may influence the optimal peptide sequences for their delivery. Herein, three peptides demonstrated effective pDNA transfection abilities. Notably, only one of the three peptides previously exhibited efficient gene-silencing effect with siRNA. These findings validate our hypothesis and offer insights for the personalized design of CPPs for the delivery of pDNA and siRNA.

Liquid foam improves potency and safety of gene therapy vectors.

Interest in gene therapy medicines is intensifying as the first wave of gene-correcting drugs is now reaching patient populations. However, efficacy and safety concerns, laborious manufacturing protocols, and the high cost of the therapeutics are still significant barriers in gene therapy. Here we describe liquid foam as a vehicle for gene delivery. We demonstrate that embedding gene therapy vectors (nonviral or viral) in a methylcellulose/xanthan gum-based foam formulation substantially boosts gene transfection efficiencies in situ, compared to liquid-based gene delivery. We further establish that our gene therapy foam is nontoxic and retained at the intended target tissue, thus minimizing both systemic exposure and targeting of irrelevant cell types. The foam can be applied locally or injected to fill body cavities so the vector is uniformly dispersed over a large surface area. Our technology may provide a safe, facile and broadly applicable option in a variety of clinical settings.

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

BACKGROUND: Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. METHODS: In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. RESULTS: Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. CONCLUSIONS: This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

Generation and Characterization of In Vitro Transcribed mRNA.

Here we describe the in vitro preparation of mRNA from DNA templates, including setting up the transcription reaction, mRNA capping, and mRNA labeling. We then describe methods used for mRNA characterization, including UV and fluorescence spectrophotometry, as well as gel electrophoresis. Moreover, characterization of the in vitro transcribed RNA using the Bioanalyzer instrument is described, allowing a higher resolution analysis of the target molecules. For the in vitro testing of the mRNA molecules, we include protocols for the transfection of various primary cell cultures and the confirmation of translation by intracellular staining and western blotting.