Transfection Reagent Research Articles

Comparison of different transient gene expression systems for the production of a new humanized anti-HER2 monoclonal antibody (Hersintuzumab).

Producing therapeutic proteins can be done quickly and on a large scale through Transient Gene Expression (TGE). Chinese hamster ovary (CHO) cell lines are commonly used to achieve this. Although there are few comparative studies, TGE has been observed in suspension-adapted CHO cells. We tested TGE's effectiveness in DG-44, CHO-S, and ExpiCHO-S cell lines with four transfection reagents. A design of experiments (DoE) was followed to optimize transfection using a recombinant monoclonal antibody (mAb) construct. To evaluate the efficacy, flow cytometry and ELISA were used. Feeding strategies and temperature shifts were implemented to enhance transfection effectiveness. The quality of the mAb was assessed through ELISA, SDS-PAGE, and proliferation inhibition assays. We adapted all cell lines to grow in suspension using a serum-free medium. Our findings from flow cytometry and ELISA tests indicate that PEI and Pmax reagents had a higher rate of transfection and mAb production than the ExpiCHO commercial transfection reagent. While DG-44 cells had better transfection efficiency than CHO-S and ExpiCHO-S, there was no significant difference between CHO-S and ExpiCHO-S. Our TGE system was more productive at 32°C than at 37°C. In the optimized TGE of Pmax-based transfection in DG-44 at 37 and 32°C, the production level of mAb was more than half of the amount of the commercial ExpiCHO-S expression system. Still, the number of transfected cells was three times higher, making it more efficient. The purified mAb from all transfected cell lines had similar structural and functional properties under different conditions. Our research shows that using Pmax and DG-44 cells in the TGE system is a cost-effective and efficient way to produce humanized monoclonal antibodies. We discovered that this method outperforms the ExpiCHO-S kit.

The cold-inducible RNA-binding protein-Thioredoxin 1 pathway ameliorates mitochondrial dysfunction and mitochondrial dynamin-related protein 1 level in the hippocampus of aged mice with perioperative neurocognitive dysfunction.

As a multi-disease model, neuroinflammation, mitochondrial dysfunction, and oxidative stress might be involved in the pathogenic process of perioperative neurocognitive dysfunction (PND). Dynamin-related protein 1 (Drp1) could mediate mitochondrial fission and play important roles in mitochondrial dynamic homeostasis and mitochondria function. The Drp1 may be involved in PND development. The cold-inducible RNA-binding protein (Cirbp) could bind to the 3'-UTR of the thioredoxin 1 (Trx1) mRNA, control oxidative stress, and improve mitochondrial function. In this study, we hypothesized that the Cirbp-Trx1 pathway could ameliorate mitochondrial dysfunction and Drp1 levels in PND mice. Differentially expressed genes were screened using the Gene Expression Omnibus (GEO) database GSE95426 and validated using PCR. Eighteen-month-old C57BL/6 mice were subjected to tibial fracture surgery to generate a PND model. Cirbp was upregulated by hippocampal stereotaxic injections of over-Cirbp plasmid according to the manufacturer's instructions for the in vivo DNA transfection reagent. Cirbp expression was measured using western blot (WB) and immunofluorescence (IF). The Morris water maze (MWM) was used to assess cognitive function. After behavioral testing, the hippocampal tissue was extracted to examine changes in mitochondrial Drp1, mitochondrial function, neuroinflammation, and oxidative stress. Differential gene screening showed that Cirbp expression was significantly downregulated (fold change >1.5, p = 0.003272) in the PND model. In this study, we also found that Cirbp protein levels were downregulated, accompanied by an impairment of cognition, a decrease in superoxide dismutase (SOD) activity, and an increase in malondialdehyde (MDA) content, mitochondrial Drp1 levels, neuroinflammation, and apoptosis. Cirbp overexpression increased Trx1 protein levels and reversed the damage. However, this protective effect was abolished by PX-12 treatment with a Trx1 inhibitor. The Cirbp-Trx1 pathway may regulate mitochondrial dysfunction and mitochondrial Drp1 expression in the hippocampus of PND mice to ameliorate cognitive dysfunction.

CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing.

Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.

Effect of REG3A on proliferation and invasion of glioma cells by regulating PI3K/Akt signaling pathway

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.

Highly reproducible and cost-effective one-pot organoid differentiation using a novel platform based on PF-127 triggered spheroid assembly.

Organoid technology offers sophisticatedin vitrohuman models for basic research and drug development. However, low batch-to-batch reproducibility and high cost due to laborious procedures and materials prevent organoid culture standardization for automation and high-throughput applications. Here, using a novel platform based on the findings that Pluronic F-127 (PF-127) could trigger highly uniform spheroid assembly through a mechanism different from plate coating, we develop a one-pot organoid differentiation strategy. Using our strategy, we successfully generate cortical, nephron, hepatic, and lung organoids with improved reproducibility compared to previous methods while reducing the original costs by 80%-95%. In addition, we adapt our platform to microfluidic chips allowing automated culture. We showcase that our platform can be applied to tissue-specific screening, such as drug toxicity and transfection reagents testing. Finally, we generateNEAT1knockout tissue-specific organoids and showNEAT1modulates multiple signaling pathways fine-tuning the differentiation of nephron and hepatic organoids and suppresses immune responses in cortical organoids. In summary, our strategy provides a powerful platform for advancing organoid research and studying human development and diseases.

Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery.

CRISPR/Cas9-based genome editing tools have enormous potential for the development of various therapeutic treatments due to their reliability and broad applicability. A central requirement of CRISPR/Cas9 is the efficient intracellular delivery of the editing machinery, which remains a well-recognized challenge, notably to deliver Cas9 in its native protein form. Herein, a phase-separating peptide with intracellular redox-triggered release properties is employed to encapsulate and deliver all three forms of CRISRP-Cas9 editing machinery, namely, pDNA, mRNA/sgRNA, and the ribonucleoprotein complex. These modalities are readily recruited within peptide coacervates during liquid-liquid phase separation by simple mixing and exhibit higher transfection and editing efficiency compared to highly optimized commercially available transfection reagents currently used for genome editing.

Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.

Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p<0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine™ 2000 and our PGC medium was the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them. This article is protected by copyright. All rights reserved.

Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization.

Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293FTM and ExpiCHO-STM with the transfection reagents ExpiFectamineTM and polyethylenimine. We discovered that there are significant differences between Expi293FTM and ExpiCHO-STM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.

A novel use of a needle-free injection system for improved nucleic acid delivery and expression in vivo.

Transfection, a nonviral method of nucleic acid delivery, often exhibits poor efficiency in vivo. The needle-based in vivo delivery of transfection reagents can be invasive. Here, we report a noninvasive protocol for in vivo gene delivery via the needle-free MED-JET H4 MULTIJET (MJH4M) device using both "home-made" glucose-based and commercial transfection reagents. The objective of this study was to compare the relative transfection efficiencies of the needle-free system to that of the needle-based delivery method. We observed a 15-fold increase in transfection efficiency using the needle-free MJH4M device when compared to the needle-based delivery method. The highest transfection efficiency was achieved using a 5% glucose solution as the delivery vehicle.

Poly(2-oxazoline)-Based Polyplexes as a PEG-Free Plasmid DNA Delivery Platform.

The present study expands the versatility of cationic poly(2-oxazoline) (POx) copolymers as a polyethylene glycol (PEG)-free platform for gene delivery to immune cells, such as monocytes and macrophages. Several block copolymers are developed by varying nonionic hydrophilic blocks (poly(2-methyl-2-oxazoline) (pMeOx) or poly(2-ethyl-2-oxazoline) (pEtOx), cationic blocks, and an optional hydrophobic block (poly(2-isopropyl-2-oxazoline) (iPrOx). The cationic blocks are produced by side chain modification of 2-methoxy-carboxyethyl-2-oxazoline (MestOx) block precursor with diethylenetriamine (DET) or tris(2-aminoethyl)amine (TREN). For the attachment of a targeting ligand, mannose, azide-alkyne cycloaddition click chemistry methods are employed. Of the two cationic side chains, polyplexes made with DET-containing copolymers transfect macrophages significantly better than those made with TREN-based copolymer. Likewise, nontargeted pEtOx-based diblock copolymer is more active in cell transfection than pMeOx-based copolymer. The triblock copolymer with hydrophobic block iPrOx performs poorly compared to the diblock copolymer which lacks this additional block. Surprisingly, attachment of a mannose ligand to either copolymer is inhibitory for transfection. Despite similarities in size and design, mannosylated polyplexes result in lower cell internalization compared to nonmannosylated polyplexes. Thus, PEG-free, nontargeted DET-, and pEtOx-based diblock copolymer outperforms other studied structures in the transfection of macrophages and displays transfection levels comparable to GeneJuice, a commercial nonlipid transfection reagent.

TECPR1 is activated by damage‐induced sphingomyelin exposure to mediate noncanonical autophagy

Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin‐like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1‐containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1‐containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage‐induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1‐ATG5‐ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1‐ATG5‐ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

Tetracycline-mediated fluorescence correlates with passage number and β-galactosidase activity in HeLa and HEK293T cells.

We report data consistent with tetracycline-mediated fluorescence having the potential to be an effective marker of senescence in immortalised cells. HeLa cells that had previously undergone more than 20 passages were transiently transfected with a plasmid encoding a novel tetracycline-inducible transgene featuring an open reading frame for green fluorescent protein. While characterising the performance of this plasmid and transfection procedure, HeLa cell fluorescence was observed to result from incubating cells with media containing 2 μg/ml tetracycline alone, without plasmid or transfection reagent. To investigate this phenomenon further, HeLa and HEK293T cells were purchased from a tissue culture collection and after cultivation over 4-23 passages, incubated with media containing 2 μg/ml tetracycline. For both cell lines, tetracycline-mediated fluorescence increase correlated with passage number increase. This effect in HeLa and HEK293T cells was also borne out by expression of β-galactosidase activity, an imperfect but widely used marker of cellular senescence. These data suggest tetracycline may have utility as a marker of cellular senescence in immortal cells and can inform future investigation and validation of this previously unreported application for this reagent.

1505-P: CD44 Knockdown in Human Adipocytes—Effect on Adipocyte Proliferation and Differentiation

Introduction: eGWAS implicates the immune-cell receptor CD44 as the top candidate gene in the molecular pathogenesis of T2D1. We have shown that in human adipose tissue, CD44 expression is associated with inflammatory gene expression, M1 macrophage phenotype, and systemic insulin resistance2. Here we sought to determine whether CD44 directly impairs adipocyte function via siRNA knockdown in human preadipocytes with quantification of proliferation, differentiation, and gene expression. Methods: Abdominal subcutaneous adipose tissue was harvested from five healthy nondiabetic obese individuals during bariatric surgery. Preadipocytes were isolated via collagenase digestion and seeded at 1×10^4 cells/well and treated with siRNA-CD44 or mock condition (control) using siRNA Maxi Transfection Reagents (Invitrogen). Cell proliferation was assayed by MTS (Promega) on Days 1, 2, 3 post transfection. Differentiation was induced at confluence (as described3), and cells were harvested at days 3, 6, 12 after confluence for gene expression and at day 12 for oil red O staining (total lipid uptake) and immunofluorescence microscopy (% differentiation = bodipy/dapi*100). Results: CD44 knockdown decreased preadipocyte proliferation and increased adipocyte differentiation and lipid uptake. Conclusion: CD44 may contribute to human insulin resistance by impairing adipocyte differentiation and fat storage. Disclosure H.Chen: None. N.R.Saini: None. E.M.Ayhan: None. T.Mclaughlin: Board Member; January, Inc., Research Support; Merck &amp; Co., Inc., Stock/Shareholder; Eiger Bio. Funding National Institutes of Health (1R21AI159024-01A1)

MicroRNA-224-5p nanoparticles balance homeostasis via inhibiting cartilage degeneration and synovial inflammation for synergistic alleviation of osteoarthritis

MicroRNAs play a crucial role in regulating cartilage extracellular matrix (ECM) metabolism and are being explored as potential therapeutic targets for osteoarthritis (OA). The present study indicated that microRNA-224-5p (miR-224-5p) could balance the homeostasis of OA via regulating cartilage degradation and synovium inflammatory simultaneously. Multifunctional polyamidoamine dendrimer with amino acids used as efficient vector to deliver miR-224-5p. The vector could condense miR-224-5p into transfected nanoparticles, which showed higher cellular uptake and transfection efficiency compared to lipofectamine 3000, and also protected miR-224-5p from RNase degradation. After treatment with the nanoparticles, the chondrocytes showed an increase in autophagy rate and ECM anabolic components, as evidenced by the upregulation of autophagy-related proteins and OA-related anabolic mediators. This led to a corresponding inhibition of cell apoptosis and ECM catabolic proteases, ultimately resulting in the alleviation of ECM degradation. In addition, miR-224-5p also inhibited human umbilical vein endothelial cells angiogenesis and fibroblast-like synoviocytes inflammatory hyperplasia. Integrating the above synergistic effects of miR-224-5p in regulating homeostasis, intra-articular injection of nanoparticles performed outstanding therapeutic effect by reducing articular space width narrowing, osteophyte formation, subchondral bone sclerosis and inhibiting synovial hypertrophy and proliferation in the established mouse OA model. The present study provides a new therapy target and an efficient intra-articular delivery method for improving OA therapy. Statement of significanceOsteoarthritis (OA) is the most prevalent joint disease worldwide. Gene therapy, which involves delivering microRNAs, has the potential to treat OA. In this study, we demonstrated that miR-224-5p can simultaneously regulate cartilage degradation and synovium inflammation, thereby restoring homeostasis in OA gene therapy. Moreover, compared to traditional transfection reagents such as lipofectamine 3000, G5-AHP showed better efficacy in both microRNA transfection and protection against degradation due to its specific surface structure. In summary, G5-AHP/miR-224-5p was developed to meet the clinical needs of OA patients and the high requirement of gene transfection efficiency, providing a promising paradigm for the future application and development of gene therapy.

TL-532, a novel specific Toll-like receptor 3 agonist rationally designed for targeting cancers: discovery process and biological characterization.

Toll-like receptor 3 (TLR3) is an innate immune receptor that recognizes double-stranded RNA (dsRNA) and induces inflammation in immune and normal cells to initiate anti-microbial responses. TLR3 acts also as a death receptor only in cancer cells but not in their normal counterparts, making it an attractive target for cancer therapies. To date, all of the TLR3-activating dsRNAs used at preclinical or clinical stages have major drawbacks such as structural heterogeneity, toxicity, and lack of specificity and/or efficacy. We conducted the discovery process of a new family of TLR3 agonists that are chemically manufactured on solid-phase support and perfectly defined in terms of sequence and size. A stepwise discovery process was performed leading to the identification of TL-532, a 70 base pair dsRNA that is potent without transfection reagent and is highly specific for TLR3 without activating other innate nucleic sensors such as RIG-I/MDA5, TLR7, TLR8, and TLR9. TL-532 induces inflammation in murine RAW264.7 myeloid macrophages, in human NCI-H292 lung cancer cells, and it promotes immunogenic apoptosis in tumor cells in vitro and ex vivo without toxicity towards normal primary cells. In conclusion, we identified a novel TLR3 agonist called TL-532 that has promising anticancer properties.

Comparison of Three Different Methods of Transfection for the Production of Recombinant Adenovirus Expressing Human Carcinoembryonic Antigen Gene.

Adenoviral vectors (AdVs) are widely used as a gene delivery vehicle and vaccine design due to their genetic stability, transfer capacity of large genes, production at high titers, and remarkable efficacy of transduction. One of the most important applications of AdVs is in cancer immunotherapy. Tumor-associated antigens are overexpressed in cancer cells; however, they cannot induce immune responses sufficiently. Therefore, the immune system must be stimulated against these antigens to kill the cancer cells. This study described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection was required. Three various transfection reagents were compared to achieve the best method of transfection. Carcinoembryonic antigen was cloned into the pAdV and transfected into the A293 cells using three different reagents, including polyethylenimine (PEI), calcium phosphate, and DMRIE-C. The PEI had the highest transfection efficiency, which was selected for the transfection of the recombinant plasmid. It has low toxicity for cells and is suitable for large-scale transfection. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA-expressing tumors.

STAT1 negatively regulates human glioma U251 cell proliferation Huang et al: Role of STAT1 in glioma

Background Glioma is one of the most malignant tumors, which leads to high mortality in cancer patients. At present, there is no effective therapy for glioma. Therefore, it is urgent and necessary to find new molecular targets for anti-glioma therapy. Objective The present study aimed to investigate the role of signal transducer and activator of transcription 1 (STAT1) in the development and progression of human glioma and related mechanisms. Methods According to the instructions of Lipofectamine TM2000 transfection reagent, we transiently transfected the plasmid pcDNA3.1-STAT1 into glioma U251 cells. Then STAT1 expression in glioma U251 and LN382 cells was detected by Western blot. MTT was performed to assay the proliferative activity of U251 cells after STAT1 transduction, flow cytometry was used to detect cell cycle and apoptosis indicators, cell migration indicator was determined by Wound healing, and Western blot was used for detecting the expression level and change trend of p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E in transfected cells. Results Overexpressed STAT1 significantly inhibited U251 cell proliferation and promoted U251 cell apoptosis. Meanwhile, high expression of STAT1 can increase the expression of p53, p21, and Caspase-8 while inhibiting the expression of bcl-2, Cyclin A, and Cyclin E. Conclusion Highly expressed STAT1 inhibits the proliferative activity of human glioma U251 cells and can promote tumor cell apoptosis and block cell cycle progression while regulating the expression of various signal transduction molecules. Thus, STAT1 has a critical function in the development and progression of glioma and is a novel target for glioma therapy.

Regulation of Baicalin on Growth of Extranodal NK/T Cell Lymphoma Cells through FOXO3/CCL22 Signaling Pathway

To investigate the effect of baicalin on the growth of extranodal NK/T cell lymphoma (ENKTCL) cells and its related mechanism. Normal NK cells and human ENKTCL cells lines SNK-6 and YTS were cultured, then SNK-6 and YTS cells were treated with 5, 10, 20 μmol/L baicalin and set control. Cell proliferation and apoptosis was detected by Edu method and FCM method, respectively, and expressions of BCL-2, Bax, FOXO3 and CCL22 proteins were detected by Western blot. Interference plasmids were designed and synthesized. FOXO3 siRNA interference plasmids and CCL22 pcDNA overexpression plasmids were transfected with PEI transfection reagent. Furthermore, animal models were established for validation. In control group and 5, 10, 20 μmol/L baicalin group, the proliferation rate of SNK-6 cells was (56.17±2.96)%, (51.92±4.63)%, (36.42±1.58)%, and (14.60±2.81)%, respectively, while that of YTS cells was (58.85±2.98)%, (51.38±1.32)%, (34.75±1.09)%, and (15.45±1.10)%, respectively. In control group and 5, 10, 20 μmol/L baicalin group, the apoptosis rate of SNK-6 cells was (5.93±0.74)%, (11.78±0.34)%, (28.46±0.44)%, and (32.40±0.37)%, respectively, while that of YTS cells was (7.93±0.69)%, (16.29±1.35)%, (33.91±1.56)%, and (36.27±1.06)%, respectively. Compared with control group, the expression of BCL-2 protein both in SNK-6 and YTS cells decreased significantly (P<0.001), and the expression of Bax protein increased in SNK-6 cells only when the concentration of baicalin was 20 μmol/L (P<0.001), while that in YTS cells increased in all three concentrations(5, 10, 20 μmol/L) of baicalin (P<0.001). The expression of FOXO3 protein decreased while CCL22 protein increased in ENKTCL cell lines compared with human NK cells (P<0.001), but the expression of FOXO3 protein increased (P<0.01) and CCL22 protein decreased after baicalin treatment (P<0.001). Animal experiments showed that baicalin treatment could inhibit tumor growth. The expression of CCL22 protein in ENKTCL tissue of nude mice treated with baicalin decreased compared with control group (P<0.01), while the FOXO3 protein increased (P<0.05). In addition, FOXO3 silencing resulted in the decrease of FOXO3 protein expression and increase of CCL22 protein expression (P<0.01, P<0.001). Baicalin can inhibit proliferation and promote apoptosis of ENKTCL cell lines SNK-6 and YTS, up-regulate the expression of Bax protein, down-regulate the expression of BCL-2 protein, and down-regulate the expression of CCL22 protein mediated by FOXO3. Animal experiment shown that the baicalin can inhibit tumor growth. Baicalin can inhibit the growth and induce apoptosis of ENKTCL cells through FOXO3/CCL22 signaling pathway.

Optimization of transfection methods for human lymphoblast TK6 cell line

Transfection has recently gained attention in the field of biomedical research due to its ability to manipulate gene expression. Every mammalian cell type has a characteristic set of requirements for optimal transfection. Some cells can be difficult to transfect and require optimization for successful transfection. Human lymphoblast TK6 cell line, an important cell line for genotoxic studies, is known to be extremely hard to transfect. Thus, optimizing transfection methods for human lymphoblast TK6 are increasingly important. To accomplish this, TK6 human lymphoblasts were transfected with plasmid constructs that expressed green fluorescent protein (GFP) and NanoLuc® activity. We compared the transfection efficiency of three commercially available transfection reagents, including Amaxa 96-well Nucleofection procedure using various solutions (SF, SE, and SG), Lipofectamine LTX, and Metafectene Pro®. The transfection efficiency and toxicity of various reagents were tested by fluorescence microscopy, luciferase activity, and cell viability assays. Amaxa 96-well Nucleofection Solution SF was identified as the best transfection reagent due to its relatively high luciferase activity, acceptable cell viability (80%), and GFP transfection efficiency (80%). Optimal conditions for transfection utilized with this reagent included 0.4 μg of plasmid DNA, 1.8 × 105 cells, and using the DS 137 Nucleofector program.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.