Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac β-myosin heavy chain (β-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Gene delivery is a complex process with several challenges when attempting to incorporate genetic material efficiently and safely into target cells. Some of the key challenges include not only efficient cellular uptake and endosomal escape to ensure that the genetic material can exert its effect but also minimizing the toxicity of the delivery system, which is vital for safe gene delivery. Of importance, if gene delivery systems are intended for biomedical applications or clinical use, they must be scalable and easy and affordable to manufacture to meet the demand. Here, we show an efficient gene delivery method using a combination of carbon dots coated by PEI through electrostatic binding to easily generate cationic carbon dots. We show a biofunctional approach to generate optimal cationic carbon dots (CCDs) that can be scaled up to meet specific transfection demands. CCDs improve cell viability and increase transfection efficiency four times over the standard of PEI polyplexes. Generated CCDs enabled the challenging transfection protocol to produce retroviral vectors via cell cotransfection of three different plasmids into packing cells, showing not only high efficiency but also functionality of the gene delivery, tested as the capacity to produce infective retroviral particles.

During triacylglycerol synthesis, the acylglycerol-3-phosphate acyltransferase (AGPAT) family catalyzes the conversion of lysophosphatidic acid to phosphatidic acid and the acylation of sn-2 fatty acids. However, the catalytic activity of different AGPAT members is different. Therefore, this study aimed to investigate the mechanism through which different AGPATs affect the efficiency of TAG synthesis and fatty acid composition. The conservation of amino acid sequences and protein domains of the AGPAT family was analyzed, and the functions of AGPAT1, AGPAT3, and AGPAT4 genes in buffalo mammary epithelial cells (BMECs) were studied using RNA interference and gene overexpression. Prediction of the protein tertiary structure of the AGPAT family demonstrated that four conservative motifs (motif1, motif2, motif3, and motif6) formed a hydrophobic pocket in AGPAT proteins, except AGPAT6. According to cytological studies, AGPAT1, AGPAT3, and AGPAT4 were found to promote the synthesis and fatty acid compositions of triacylglycerol, especially UFA compositions of triacylglycerol, by regulating ACSL1, FASN, GPAM, DGAT2, and PPARG gene expression. This study provides new insights into the role of different AGPAT gene family members involved in TAG synthesis, and a reference for improving the fatty acid composition of milk.

Gene therapy is extensively studied as a realistic and promising therapeutic approach for treating inherited and acquired diseases by repairing defective genes through introducing (transfection) the \"healthy\" genetic material in the diseased cells. To succeed, the proper DNA or RNA fragments need efficient vectors, and viruses are endowed with excellent transfection efficiency and have been extensively exploited. Due to several drawbacks related to their use, nonviral cationic materials, including lipidic, polymeric, and dendrimer vectors capable of electrostatically interacting with anionic phosphate groups of genetic material, represent appealing alternative options to viral carriers. Particularly, dendrimers are highly branched, nanosized synthetic polymers characterized by a globular structure, low polydispersity index, presence of internal cavities, and a large number of peripheral functional groups exploitable to bind cationic moieties. Dendrimers are successful in several biomedical applications and are currently extensively studied for nonviral gene delivery. Among dendrimers, those derived by 2,2-bis(hydroxymethyl)propanoic acid (b-HMPA), having, unlike PAMAMs, a neutral polyester-based scaffold, could be particularly good-looking due to their degradability in vivo. Here, an overview of gene therapy, its objectives and challenges, and the main cationic materials studied for transporting and delivering genetic materials have been reported. Subsequently, due to their high potential for application in vivo, we have focused on the biodegradable dendrimer scaffolds, telling the history of the birth and development of b-HMPA-derived dendrimers. Finally, thanks to a personal experience in the synthesis of b-HMPA-based dendrimers, our contribution to this field has been described. In particular, we have enriched this work by reporting about the b-HMPA-based derivatives peripherally functionalized with amino acids prepared by us in recent years, thus rendering this paper original and different from the existing reviews.

Biomaterial-mediated, spatially localized gene delivery is important for the development of cell-populated scaffolds used in tissue engineering. Cells adhering to or penetrating into such a scaffold are to be transfected with a preloaded gene that induces the production of secreted proteins or cell reprogramming. In the present study, we produced silica nanoparticles-associated pDNA and electrospun scaffolds loaded with such nanoparticles, and studied the release of pDNA from scaffolds and cell-to-scaffold interactions in terms of cell viability and pDNA transfection efficacy. The pDNA-coated nanoparticles were characterized with dynamic light scattering and transmission electron microscopy. Particle sizes ranging from 56 to 78 nm were indicative of their potential for cell transfection. The scaffolds were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, stress-loading tests and interaction with HEK293T cells. It was found that the properties of materials and the pDNA released vary, depending on the scaffold\'s composition. The scaffolds loaded with pDNA-nanoparticles do not have a pronounced cytotoxic effect, and can be recommended for cell transfection. It was found that (pDNA-NPs) + PEI9-loaded scaffold demonstrates good potential for cell transfection. Thus, electrospun scaffolds suitable for the transfection of inhabiting cells are eligible for use in tissue engineering.

BACKGROUND: We report on a large family of Chinese Han individuals with hidrotic ectodermal dysplasia (HED) with a variation in GJB6 (c.31G>A). The patients in the family had a triad of clinical manifestations of varying degrees. Although the same variation locus have been reported, the clinical manifestations of this family were difficult to distinguish from those of congenital thick nail disorder, palmoplantar keratosis, and congenital hypotrichosis.
METHODS: This investigation involved a large Chinese family of 46 members across five generations and included 12 patients with HED. The proband (IV4) was a male patient with normal sweat gland function and dental development, no skeletal dysplasia, no cognitive disability, and no hearing impairments. His parents were not consanguineously married. Physical examination of the proband revealed thinning hair and thickened grayish-yellow nails and toenails with some longitudinal ridges, in addition to mild bilateral palmoplantar hyperkeratosis. GJB6, GJB2, and GJA1 have been reported to be the causative genes of HED; therefore, we subjected the patient\'s samples to Sanger sequencing of these three genes. In this family, the variation locus was at GJB6 (c.31G>A, p.Gly11Arg). Overexpression vectors of wild-type GJB6 and its variants were established and transfected into HaCaT cell models, and the related mRNA and protein expression changes were determined using real-time reverse transcriptase-polymerase chain reaction and Western blot, respectively.
CONCLUSIONS: We report another HED phenotype associated with GJB6 variations, which can help clinicians to diagnose HED despite its varying presentations.

OBJECTIVE: This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery.
METHODS: Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) with various drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) were determined, and selected powders were used for further characterization.
RESULTS: Of the nine dry powders prepared, their MMAD values were about 1-2 µm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before TFF. Powders prepared with Tris-EDTA buffer or cosolvents (i.e., 1,4-dioxane or tert-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without Tris-EDTA were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix structure, with a moisture content of < 2% (w/w). Agarose gel electrophoresis confirmed the chemical integrity of the pDNA after it was subjected to TFF and after the TFF powder was actuated. A cell transfection study confirmed that the activity of the pDNA did not change after it was subjected to TFF.
CONCLUSIONS: It is feasible to use TFF to produce aerosolizable pDNA dry powder for pulmonary delivery, while preserving the integrity and activity of the pDNA.

Cell transfection efficiency is still a limiting factor in gene function research. A method that allows isolation and enrichment of the transfection-positive cells is an effective solution. Here, we report a transfection-positive cell sorting system that utilizes GPI-anchored GST (Glutathione S-transferase) as a plasmid marker. The Glutathione S-transferase fusion protein will be expressed and displayed on the cell surface through GPI anchor, and hence permits the positive cells to be isolated using Glutathione (GSH) Magnetic Beads. We prove that the system works efficiently in both the adherent Lenti-X 293T cells and the suspension K-562 cells. The affinity cell sorting procedure efficiently enriched positive cells from 20% to 98% in K-562 cells. The applications in gene knockdown and overexpression experiments in K-562 cells dramatically enhanced the extent of gene alteration, with the gene knockdown efficiency increasing from 7% to 60% and the gene overexpression level rising from 47 to 253 times. This Glutathione S-transferase affinity transfection-positive cell sorting method is simple and fast to operate, large-instrument free, low cost, and hence possesses great potential in gene function study in vitro.

Transfection is the process by which nucleic acids are introduced into eukaryotic cells. This is fundamental in basic research for studying gene function and modulation of gene expression as well as for many bioprocesses in the manufacturing of clinical-grade recombinant biologics from cells. Transfection efficiency is a critical parameter to increase biologics\' productivity; the right protocol has to be identified to ensure high transfection efficiency and therefore high product yield. Design of experiments (DoE) is a mathematical method that has become a key tool in bioprocess development. Based on the DoE method, we developed an operational flow that we called \"Design of Transfections\" (DoT) for specific transfection modeling and identification of the optimal transfection conditions. As a proof of principle, we applied the DoT workflow to optimize a cell transfection chemical protocol for neural progenitors, using polyethyleneimine (PEI). We simultaneously varied key influencing factors, namely concentration and type of PEI, DNA concentration, and cell density. The transfection efficiency was measured by fluorescence imaging followed by automatic counting of the green fluorescent transfected cells. Taking advantage of the DoT workflow, we developed a new simple, efficient, and economically advantageous PEI transfection protocol through which we were able to obtain a transfection efficiency of 34%.

Nano-SiO2 is increasingly used in diagnostic and biomedical research because of its ease of production and relatively low cost and which is generally regarded as safe and has been approved for use as a food or animal feed ingredient. Although recent literature reveals that nano-SiO2 may present toxicity and DNA damage, however, the underlying mechanism remains poorly understood. Since in previous studies, we found that nano-SiO2 treatment down-regulated the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells and PAPR-1 knockdown can aggravate DNA damage induced by nano-SiO2. Therefore, we speculate whether PARP-1 overexpression can protect DNA from damage induced by nano-SiO2. However, our data demonstrated that overexpression of PARP-1 in HaCaT cells slightly enhanced the cellular proliferation of undamaged cells, when compared with both empty vector control cells and parental cells, but had drastic consequences for cells treated with nano-SiO2. The PARP-1 overtransfected cells were sensitized to the cytotoxic effects and DNA damage of nano-SiO2 compared with control parental cells. Meanwhile, flow cytometric analysis of nano-SiO2 stimulated poly(ADP-ribose) synthesis revealed consistently larger fractions of cells positive for this polymer in the PARP-1 overexpression cells than in control clones. Combining our previous research on PARP-1 knockdown HaCaT cells, we hypothesize that an optimal level of cellular poly(ADP-ribose) accumulation exists for the cellular recovery from DNA damage.