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The CRISPR/Cas9 system has been harnessed to cleave a targeted DNA fragment via its Cas nuclease activity under the direction of guide RNA for rendering gene insertions, deletions, and point mutations in basic research and clinical applications. There are a number of vehicles, including lipofectamine, viruses, and nanoparticles, that can deliver the CRISPR/Cas9 system, but all these methods face numerous challenges during their application in life science contexts. Here, we focus on the delivery of CRISPR/Cas9 via nanoparticles because this method has shown great advantages in terms of safety, simplicity and flexibility.

Background and aims: Human interferon beta-1a (hIFNβ-1a) is a 22.5-kDa glycoprotein used to treat diseases such as multiple sclerosis (MS). Because of appropriate post-translation modifications, protein isolation, and lack of toxicity in Chinese hamster ovary (CHO) cells, we cloned hIFNβ-1a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinant protein. Methods: The hIFNβ-1a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and then syntactically constructed in the pUC57 vector. After confirmation, the synthesized sequence was cloned into the pcDNA3.1 expression vector by using EcoRI and XhoI sites via Escherichia coli DH5α competent cells. Then, the recombinant vector pcDNA-hHIFNβ1a was linearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened by gentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinant protein expression was qualitatively evaluated using different techniques. Results: The hIFNβ1a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ1a-pcDNA3.1 in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfected CHO cells was performed using gentamicin. The protein expression was confirmed by RT-PCR, MTT assay, SDS-PAGE, and Western blot. Comparison of the optimized and control samples demonstrated that chemical treatment enhanced the protein expression. Conclusion: We achieved the stable clones of CHO cells expressing the active form of human interferon beta.

The present study was carried out to silence the transcription factor genes ZCT1, ZCT2 and ZCT3 via lipofectamine based antisense LNA GapmeRs transfection into the protoplasts of established photomixotrophic cell suspensions. The photomixotrophic cell suspensions with a threshold of 0.5% sucrose were raised and established using two-tiered CO2 providing flasks kept under high light intensity. The photomixotrophic cell suspensions showed morphologically different thick-walled cells under scanning electron microscopic analysis in comparison to the simple thin-walled parenchymatous control cell suspensions. The LC–MS analysis registered the vindoline production (0.0004 ± 0.0001 mg/g dry wt.) in photomixotrophic cell suspensions which was found to be absent in control cell suspensions. The protoplasts were isolated from the photomixotrophic cell suspensions and subjected to antisense LNA GapmeRs silencing. Three lines, viz. Z1A, Z2C and Z3G were obtained where complete silencing of ZCT1, ZCT2 and ZCT3 genes, respectively, was observed. The Z3G line was found to show maximum production of vindoline (0.038 ± 0.001 mg/g dry wt.), catharanthine (0.165 ± 0.008 mg/g dry wt.) and vinblastine (0.0036 ± 0.0003 mg/g dry wt.). This was supported by the multifold increment in the gene expression of TDC, SLS, STR, SGD, d4h, dat, CrT16H and Crprx. The present work indicates the master regulation of ZCT3 knockdown among all three ZCTs transcription factors in C. roseus to enhance the terpenoid indole alkaloids production. The successful silencing of transcription repressor genes has been achieved in C. roseus plant system by using photomixotrophic cell cultures through GapmeR based silencing. The present study is a step towards metabolic engineering of the TIAs pathway using protoplast transformation in C. roseus.

Most oligonucleotides fail to enter a cell and cannot escape from endosomes after endocytosis because of their negative charge and large molecular weight. More efficient cellular delivery of oligonucleotides should be developed for the widespread implementation of antisense imaging. The purpose of this study was to construct a novel antisense nanoprobe, 99mTc-labeled anti-miRNA oligonucleotides/cell-penetrating peptide PepFect6 (99mTc-AMO/PF6), and to evaluate its efficacy for imaging the miRNA-21 expression in A549 lung adenocarcinoma xenografts. Naked AMO and commercial Lipofectamine 2000-based nanoparticles (AMO/LIP) were used for comparison. The cellular delivery efficiency of AMO/PF6 was first investigated by laser confocal scanning microscopy using Cy5.5-labeled probes and further validated by in vivo fluorescence imaging. Then, the probes were labeled with 99mTc via hydrazinonicotinamide (HYNIC). The cytotoxicity assay, cellular uptake, and retention kinetics of the probes were evaluated in vitro. The biodistribution of the probes was investigated in A549 lung cancer xenografts, and SPECT imaging was performed in vivo. AMO/PF6 showed lower cytotoxicity than AMO/LIP (P < 0.05) but showed no significant difference with naked AMO. Fluorescence microscopy demonstrated more extensive and scattered signal distribution inside the A549 cells by AMO/PF6 than AMO/LIP. The labeling efficiency of 99mTc-AMO/PF6 was 72.6 ± 1.42%, and the specific activity was 11.6 ± 0.13 MBq/ng. The cellular uptake of 99mTc-PF6/AMO peaked at 12 h, with the uptake of 11.24 ± 0.12 mol/cell × 10-16, and the cellular retention of 99mTc-AMO/PF6 was 3.92 ± 0.15 mol/cell × 10-16 at 12 h after interrupted incubation. AMO/PF6 showed higher cellular uptake and retention than naked AMO and AMO/LIP. The biodistribution study showed that the tumor had the highest radioactivity accumulation, with the uptake ratio of tumor/muscle (T/M) increasing from 14.59 ± 0.67 to 21.76 ± 0.98 between 1 and 6 h after injection, followed by the uptake in the kidneys and the liver. The results of in vivo fluorescence and SPECT imaging were consistent with the results of the biodistribution. The tumor was visualized at 6 h after injection of AMO/PF6 with the highest T/M ratio among these probes (P < 0.05). PF6 improves cellular delivery of antisense oligonucleotides via noncovalent nanoparticles. 99mTc-AMO/PF6 shows favorable imaging properties and is promising for miRNAs imaging in vivo.

Endothelial dysfunction is involved in various aspects of vascular biology and different stages of cardiovascular diseases (CVDs). Nucleotide-binding oligomerization domain-containing protein (NOD) 2, a pivotal innate immune receptor for muramyl dipeptide (MDP), has been reported to be a central regulator in CVDs. Previously, we reported that NOD2 played a leading role in MDP-triggered oxidative stress in endothelial cells (ECs). However, whether NOD2 participates in the regulatory mechanism of vascular cell adhesion molecule‑1 (VCAM-1) and endothelin‑1 (ET-1) expression was not elucidated.Human umbilical vein endothelial cells (HUVECs) were stimulated with MDP for 12 h. mRNA expression of VCAM‑1 and ET‑1 was detected using real time polymerase chain reaction (PCR). Scrambled control small interfering RNA (siRNA) and NOD2 siRNA were transfected into HUVECs using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA). Furthermore, pyrrolidine dithiocarbamate was adopted to investigate the effect of nuclear factor κB (NF-κB) on NOD2-mediated VCAM‑1 and ET‑1 gene expression in MDP-treated HUVECs.Data showed that MDP significantly increased VCAM‑1 and ET‑1 mRNA expression, which was dependent on NOD2. In addition, NF-κB inhibition suppressed NOD2-mediated gene expression of VCAM‑1 and ET‑1.Collectively, we confirmed NOD2 aggravated VCAM‑1 and ET‑1 gene expression through NF-κB in HUVECs treated with MDP.ZIELSETZUNGEN: Eine endotheliale Dysfunktion ist an vielfältigen Aspekten der Gefäßbiologie und an unterschiedlichen Stadien von Herz-Kreislauf-Erkrankungen (CVDs) beteiligt. NOD2 („nucleotide-binding oligomerization domain-containing protein“), ein entscheidender angeborener Immunrezeptor für Muramyldipeptid (MDP), wurde als zentraler Regulator bei Herz-Kreislauf-Erkrankungen beschrieben. In früheren Veröffentlichungen haben wir dargelegt, dass NOD2 eine wesentliche Funktion beim MDP-getriggerten oxidativen Stress in Endothelzellen (ECs) hat. Ob NOD2 jedoch am Regulationsmechanismus der Expression von VCAM‑1 („vascular cell adhesion molecule-1“) und ET‑1 („endothelin-1“) beteiligt ist, ließ sich nicht eindeutig klären.Humane Nabelvenenendothelzellen (HUVECs) wurden 12 h lang mit MDP stimuliert. Wir konnten die mRNA-Expression von VCAM‑1 und ET‑1 mittels Real-time-PCR (Polymerasekettenreaktion) nachweisen. „Scrambled control“ siRNA („small interfering RNA“) und NOD2-siRNA wurden mit Lipofectamine 2000-Reagenz (Invitrogen, Waltham/MA, USA) in HUVECs transfiziert. Ferner wurde Pyrrolidindithiocarbamat eingesetzt, um die Wirkung des nukleären Faktors κB (NF-κB) auf die NOD2-vermittelte VCAM-1- und ET-1-Genexpression in MDP-vorbehandelten HUVECs zu untersuchen.Die Befunde zeigten, dass MDP die von NOD2 abhängige VCAM-1- und ET-1-mRNA-Expression signifikant erhöhte. Darüber hinaus supprimerte die NF-κB-Inhibition die NOD2-vermittelte Genexpression von VCAM‑1 und ET‑1.Insgesamt bestätigten wir die durch NOD2 beeinträchtigte VCAM-1- und ET-1-Genexpression mittels NF-κB in mit MDP vorbehandelten HUVECs.

MicroRNA (miR)-518-3p has been shown to function as a tumor suppressor. This study was conducted to investigate the effects of miR-518-3p in colorectal cancer (CRC). The miR-518-3p mimic, mimic negative control (NC), miR-518-3p inhibitor, inhibitor-NC, ShRNA-TRIP4, and ShRNA-NC vectors were transfected into SW480 cells using Lipofectamine 2000. Cell viability was detected using CCK-8. Colony formation, cell invasiveness, and cell migration were assessed by plate colony formation, Transwell assays, and wound healing assays, respectively. Relative mRNA and protein levels were detected using RT–qPCR and Western blot, respectively. The target gene thyroid hormone receptor interactor 4 (TRIP4) of miR-518-3p was identified and further verified using dual-luciferase reporter assay. Compared with normal tissues, levels of miR-518-3p were decreased and TRIP4 was significantly increased in the tissues from patients with CRC. Following transfection with a miR-518-3p mimic or ShRNA-TRIP4, cell viability decreased in a time-dependent manner, and colony formation rate, wound closure rate, and the number of invasive cells were much lower for the transfected cells than in the corresponding NC and control groups. miR-518-3p overexpression or silencing of TRIP4 significantly down-regulated the expression of MMP-2 and MMP-9. Knockdown of miR-518-3p had the opposite effects, and TRIP4 was identified as a target of miR-518-3p. The inhibitory effects of miR-518-3p on the progressions of CRC are associated with TRIP4.

The study emphasized on the development of an efficient, receptor-targeted non-viral gene delivery vehicle for gene therapy of triple negative breast cancer (TNBC). Here, naturally abundant guar gum based non-viral carrier was developed through conjugating by low molecular weight polyethylenimine (LPEI) (GNP) using napthalic anhydride coupling agent and characterized them by FT-IR, 1H NMR, XRD and UV spectrophotometer. The carrier was found to be cytocompatible as revealed by MTT assay against MDA-MB-231 and HeLa cell lines and excellent blood compatibility till the concentration of 200 μg/ml. In addition to these, the carrier exhibited excellent gene binding capability and formed spherical shaped complexes. The carrier showed very high in vitro transfection efficiency in TNBC cell (MDA-MB-231) compared to lipofectamine 2000 (LF2k) which could be justified by its high buffering capacity. Therefore, GNP may be an attractive non-viral gene carrier for gene therapy of TNBC in future.

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Background & Objective: Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from various mutations in B or T-lymphoid progenitors. MicroRNAs (miRNAs) regulate gene expression by binding to the 3' untranslated region of protein-coding genes. Dysregulation of miRNA expression may result in the development of cancerous phenotypes. Therefore, for the first time in this field, the present study aims to investigate the effect of overexpression of miR-506 in Jurkat (acute T cell leukemia) cell line. Methods: In this study, Jurkat cell lines were cultured in RPMI-1640 medium. Next, miR-506 was transfected with concentrations of 50 and 100 nM with Lipofectamine 2000. The accuracy of the transfection was confirmed by the transfection of siRNA conjugated with FITC. 48 h after transfection, the cells were prepared for other tests (flow cytometry, MTT assay, and RNA extraction). The expression level of miR-506 in the cells was analyzed using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Finally, SPSS 21 software was used for the data analysis. Results: According to our results, the viability of cells in concentrations of 50 and 100 nM was significantly higher than the control group. By overexpression of miR-506, the expressions of pro-apoptotic genes (p53, p21) and anti-apoptotic gene B-cell lymphoma-2 (BCL-2) are decreased and increased, respectively. Conclusion: This study showed that miR-506 may function as an oncogenic miRNA in the T- ALL cell line. In conclusion, overexpression of miR-506 leads to an increase in viable cancer cells.

Transfection of surface adherent cells remain as a standard methodology for lentiviral production for early phase clinical studies and research purposes. Production today is based on transient co-transfection of three or four plasmids, where the viral elements are encoded separately for safety reasons. Assembly of functional lentiviral particles requires all plasmids to be efficiently transfected into each cell, a notable challenge with many currently available methods for transient transfection. We have previously demonstrated the significant improvement of cationic polymer-based transfection in various cell types using a combination of fusogenic lipids and histone deacetylase 6 inhibitor (Enhancers). In this report, we focused on the transfection step and the feasibility of improving lentiviral production using the Enhancers. After optimization of DNA amount and N/P ratio, transfection using seven commercial gene carriers showed comparable maximal efficiency of production with high cell viability. In the presence of Enhancers, the production of functional lentivirus using LPEI was increased by as much as tenfold and outperformed lentiviral production using Lipofectamine 3000. We demonstrate a scalable and optimized workflow where the use of the Enhancers significantly improved the lentiviral particle production in various HEK293 cell lines.

Objective Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications. Materials and methods CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 μL/g). Results Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours. Conclusion The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ± 4.56%, P

A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.

Background Producing an appropriate number of engineered cells is considered as one of the influential factors in the successful treatments with chimeric antigen receptor (CAR) T cells. To this aim, the transduction rate of the viral vectors can play a significant role. In addition, improving transduction rates can affect the success rate of this treatment due to hard-transduced T lymphocytes. Results In this study, activated T cells were transduced using different transduction methods such as spinoculation, retronectin, polybrene, spinoculation + retronectin, and spinoculation + polybrene after selecting the most efficient transfection method to produce recombinant viral particles containing MUC1 CAR. PEI and lipofectamine with the amount of 73.72 and 72.53%, respectively, showed the highest transfection rates with respect to calcium phosphate (14.13%) for producing lentiviral particles. However, the cytotoxicity of transfection methods was not significantly different. Based on the results, spinoculation + retronectin leads to the highest transduction rates of T cells (63.19 ± 4.45%) relative to spinoculation + polybrene (34.6 ± 4.44%), polybrene (10.23 ± 0.79%), retronectin (10.37 ± 1.85%), and spinoculation (21.11 ± 1.55%). Further, the polybrene (40.02%) and spinoculation + polybrene (48.83% ± 4.83) increased cytotoxicity significantly compared to other groups. Conclusion Improving transduction conditions such as using spinoculation with retronectin can ameliorate the production of CAR-T cells by increasing the rate of transduction, as well as the success rate of treatment.

The end result of a variety of cardiovascular diseases is heart failure. Heart failure patients’ morbidity and mortality rates are increasing year after year. Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (HucMSC-EVs) have recently been discovered to be an alternative treatment for heart failure, according to recent research. In this study, we aimed to explore the underlying mechanisms in which HucMSC-EVs inhibited doxorubicin (DOX)-induced heart failure in AC16 cells. An miR-100-5p inhibitor and an miR-100-5p mimic were used to transfect HucMSCs using Lipofectamine 2000. HucMSC-EVs were isolated and purified using the ultracentrifugation method. AC16 cells were treated with DOX combined with HucMSC-EVs or an EV miR-100-5-p inhibitor or EV miR-100-5-p mimic. ROS levels were measured by a flow cytometer. The levels of LDH, SOD, and MDA were measured by biochemical methods. Apoptotic cells were assessed by a flow cytometer. Cleaved-caspase-3 and NOX4 protein expression were determined by Western blot. The experiment results showed that HucMSC-EVs inhibited DOX-induced increased levels of ROS, LDH, and MDA, and decreased levels of SOD which were reversed by an EV miR-100-5-p inhibitor, while EV miR-100-5-p mimic had a similar effect to HucMSC-EVs. At the same time, HucMSC-EV-inhibited DOX induced the increases of apoptotic cells as well as NOX4 and cleaved-caspase-3 protein expression, which were reversed by an EV miR-100-5-p inhibitor. Furthermore, the NOX4 expression was negatively regulated by miR-100-5p. Overexpression of NOX4 abolished the effects in which HucMSC-EVs inhibited DOX-induced ROS, oxidative stress, and apoptosis increases. In conclusion, these results indicate that HucMSC-EVs inhibit DOX-induced heart failure through the miR-100-5p/NOX4 pathway.

Due to its small size, easy accessibility and immune privileged environment, the eye represents an ideal target for therapeutic nucleic acids in the treatment of posterior segment ocular diseases, such as age-related macular degeneration (AMD). Among nanocarriers that can be used to achieve nucleic acid delivery, virus-like particles (VLPs) obtained from the Cowpea chlorotic mottle virus (CCMV) are an appealing platform, because of their loading capacity, ease of manufacture and amenability for functionalization. Herein, antisense oligonucleotide-loaded CCMV nanoparticles, intended for intravitreal injection, are evaluated for selective silencing of miR-23, an important target in AMD. CCMV nanoparticles loaded with anti-miR-23 locked nucleic acid and stabilized using the 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) cross-linker, are assembled in vitro with a loading efficiency up to 80%. VLPs are found to be stable at 37 °C in the vitreous humor up to 24 hours. Nanoparticle cytotoxicity, cellular uptake and transfection efficacy are evaluated in endothelial cells. Selective miRNA down-regulation is achieved by the loaded CCMV VLPs both in absence and presence of Lipofectamine, with efficacies of ≈40% and more than 80%, respectively. The authors' findings pave the way for the future development of CCMV nanoparticles as oligonucleotide delivery platform to treat posterior segment ocular diseases.

A new continuous fish cell line (CAM) has been successfully derived from the muscle tissues of grass goldfish, Carassius auratus. The primary cell cultures were initiated by incomplete trypsinization first and then explant culture in a Leibovitz-15 medium supplemented with 15% fetal bovine serum and 10% fish muscle extract. It was found that the CAM cells were very sensitive to trypsinization and needed to be sub-cultured at a low trypsin concentration of 0.0625% to be able to go through the crisis of spontaneous immortalization transformation, and afterward a total of five derivative cell strains were isolated from the original CAM cell line. This spontaneous immortalization transformation event was recorded successively at passages 44-47, beginning with a large-scale apoptosis and senescence and followed by mitosis arrest and re-activation, thus designated as cell strain CAM-44A, 44B, 45A, 44B, and 47A. Now both the CAM cell line and strains had been sub-cultured for more than 89 times and could be well cryopreserved in the growth medium containing 5% dimethylsulfoxide. Chromosome analysis and COI gene analysis had confirmed the grass goldfish origin of these CAM cells. Transfection potential analysis indicated that Lipofectamine LTX and Xfect were two suitable transfection reagents to be used in the gene delivery of CAM cells with a transfection efficiencies up to 11±6% and 8±3% in the CAM cell lines, respectively. Among the five cell strains, CAM-47A showed the highest transfection potential with a transfection efficiency up to 28 ± 5%. This work will provide a useful cell source for works on the cell-based artificial fish meat production and functional studies of fish myogenesis-related genes.

BACKGROUND Our primary aim of the current study was to explore the correlation between plasma CRABP2 and migration, proliferation and invasion of non-small cell lung cancer (NSCLC) cells. METHODS Human lung cancer cell line A549 was used in the present study, which was cultured in 6-well plates (1 × 106 cells/well) and then transfected with pcDNA-CRABP2 and pcDNA, siRNA with the use of Lipofectamine 2000 based on the manufacturer's protocol. The expression of CRABP2 mRNA was detected through real-time PCR. Proliferation was further detected using MTT assays, and apoptosis was monitored and recorded with the application of flow cytometry. The expression of E-cadherin, MMP9, vimentin and related pathway proteins was detected by Western blotting assays. Transwell assays and cell scratch assays were utilized for the detection of migration and invasion ability of A549 cells. RESULTS RT-PCR results showed The CRABP2 mRNA transcript levels in the CRABP2 overexpression group were higher when comparing those of the empty vector group (P < 0.05). By MTT assays, CRABP2 overexpression promoted cellular proliferation, while CRABP2 downregulation inhibited cellular proliferation. CRABP2 overexpression inhibited cell apoptosis and promoted cellular proliferation. The number of TUNEL staining positive cells was the lowest in the CRABP2 overexpression group, and the siRNA transfection group had increased apoptosis. CRABP2 downregulation reduced EMT in cells and cell migration and invasion reflected from western blotting results and cell migration and invasion assay results, respectively. CONCLUSION Inhibition of plasma CRABP2 expression offers the potential in terms of reducing the expression of MAPKs and proteins in the NF-κB pathway and inhibiting the proliferation and migration of NSCLC cells, which is ideally suited for further treatment for NSCLC.

One of the most popular nonviral delivery systems for gene therapy constructs are carriers based on polyethylenimine (PEI) DNA complexes. A number of disadvantages associated with the lack of targeted delivery and increased cytotoxicity are overcome by adding auxiliary molecules to the complexes. An example of this is chondroitin sulfate (CS). The purpose of this work was to assess the effect of CS on the transfection properties of DNA–PEI complexes under different conditions of their preparation and transfection protocols. All complexes were prepared in solutions with high and low ionic strength. Transfection of C26 cells was performed according to two protocols differing in the presence of serum in the medium. The portion of transfected cells, transgene expression level, and cell viability were the main parameters of assessing the transfection efficiency. In binary DNA–PEI complexes prepared in different salt conditions, using different transfection protocols, the difference in the portion of transfected cells reached ten times. Addition of CS improved this transfection efficiency indicator up to 6.5 times, while the maximum difference in this indicator for the corresponding ternary complexes was reduced to 2.5 times. Changes in the proportion of CS in the composition of the complexes had an insignificant effect on their transfection properties. In the case of complexes prepared in high ionic strength solutions, the order of CS addition was also important. The best results of transfection efficiency were achieved with ternary complexes prepared in low ionic strength solutions, using a serum-free protocol, while these indicators were comparable with the data for Lipofectamine 2000. The addition of chondroitin sulfate improves the transfection properties of DNA–PEI complexes and makes them less dependent on the methods of preparation and transfection.

To investigate the effect of microRNA-101-3p (miRNA-101-3p) on proliferation, invasion and apoptosis of gastric cancer (GC) cells, and to explore its influence mechanism. Human GC cell line (AGS) and normal human gastric epithelial cell line (GES-1) were used in this study. The cells were transfected with proto-oncogene serine/threonine-protein kinase (PIM 1) overexpression plasmid, miRNA-101-3p mimics, or miRNA-101-3p non-homologous sequence using lipofectamine 2000. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the expressions of miRNA-101-3p and PIM 1 in GC cells. Cell counting kit-8 (CCK-8) and Transwell assays were used to assess the effect of miRNA-101-3p on proliferation and invasion of GC cells. The regulatory effect of miRNA-101-3p on PIM 1 was assessed using bioinformatics analysis and luciferase reporter gene assay. The expression of miRNA-101-3p was significantly down-regulated in GC cells, relative to normal human gastric epithelial cells (p < 0.05). However, the expression of PIM 1 mRNA was significantly upregulated in GC cells, when compared with normal gastric epithelial cells (p < 0.05). The expression level of miRNA-101-3p was significantly higher in miRNA-101-3p mimic group than in miRNA-101-3p control and normal control groups (p < 0.05). Cell proliferation in miRNA-101-3p mimic group was significantly and time-dependently reduced, when compared with miRNA-101-3p control and normal control groups (p < 0.05). Transfection with miRNA-101-3p mimics significantly increased the invasiveness of AGS cells, and significantly promoted their apoptosis (p < 0.05). Results of qRT-PCR and Western blotting showed that increased miRNA-101-3p expression significantly reduced the expression of PIM 1, while decreased miRNA-101-3p expression promoted the expression of PIM 1 (p < 0.05). Results of bioinformatics showed that miRNA-101-3p had specific binding sequence in the 3'UTR region of PIM 1. Cloning of miRNA-101-3p sequence into the luciferase reporter plasmid led to significant inhibition of the expression of PIM 1 (p < 0.05), but had no inhibitory effect on mutated PIM 1 3'UTR (p > 0.05). Overexpression of PIM 1 significantly reversed the inhibition of proliferation and invasion, and promotion of apoptosis by miRNA-101-3p (p < 0.05). These results indicate that miRNA-101-3p inhibits the proliferation and invasion of GC cells, and promotes their apoptosis by regulation of the expression of PIM-1.

Background/aims Fibroblast growth factor 21 (FGF21) plays a protective role in ischemia/reperfusion induced cardiac injury. However, the exact molecular mechanism of FGF21 action remains unclear. This study was designed the protective effect of FGF21 on the heart and its mechanism. Method Adenovirus vector expressing FGF21 or control β-galactosidase was injected into the myocardium of mice. Myocardial injury was observed by tissue staining and immunohistochemical staining. The expression level of caspases-3 and galectin-3 in myocardial cells were observed by immunoblotting. Then, hypoxia-induced cell model was established. Small interfering RNA (SiRNA) and plasmid were transfected into H9c2 using Lipofectamine 2000 reagent (Invitrogen). The expression levels of galectin-3, ECM and cystatin-3 in cells were observed by immunoblotting, and the relationship between fibroblast growth factor 21 and galectin-3 was analyzed. Result Cell test in vitro showed that FGF21 could inhibit apoptosis and decrease the expression of ECM (ColIaI, fibronectin, and alpha-SMA) under hypoxia. Western blot data showed that hypoxia-induced cell damage increased galectin-3 levels, while FGF21 decreased galactose lectin-3 levels. In addition, inhibition of galactose agglutinin-3 expression by siRNA enhanced the cardioprotective effect of FGF21, while overexpression of galectin-3 reduced the cardioprotective effect of fibroblast growth factor 21. Conclusion FGF21 may be a novel therapy for hypoxia-induced cardiac injury by regulating the expression of galectin-3.

Effect of Runx3 gene on the cell proliferation and invasion of rectal cancer was investigated to explore potential new targets for targeted treatment of rectal cancer. The Runx3 overexpression group (OE group), blank plasmid control group, negative control and blank group of the rectal cancer HRC-9698 cell strain were set. The overexpressed Runx3 plasmid was transfected in OE group; the empty plasmid was transfected in blank plasmid control group; only liposome Lipofectamine was added to negative control group; only 1640 medium was used in blank group. RT-qPCR was used for detection of the mRNA expression of Runx3 in different groups; CCK-8 kit for detection of cell proliferation in different groups; Transwell chamber test for detection of cell strain invasion in different groups. The mRNA expression of Runx3 gene in OE group was the highest, significantly higher than that in blank plasmid control group, negative control and blank group (P

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

Background MicroRNAs (miRNAs) have been reported to play crucial roles in cancer cell processes, including proliferation, metastasis and cell cycle progression. We aimed to identify miRNAs that could act as suppressors of cell growth and invasion in non-small cell lung cancer (NSCLC). Methods Fifteen paired NSCLC tissue samples and pericarcinomatous normal tissues were collected and preserved in liquid nitrogen. The expression levels of miR-340-5p and ZNF503 mRNA were detected using a qPCR assay. The transfection of plasmids was conducted using Lipofectamine 3000 according to the manufacturer’s protocol. Cell proliferation was determined using a CCK-8 assay. The protein levels of endothelial–mesenchymal transition markers were measured using a western blot assay. Cell invasive ability was evaluated using a transwell assay. TargetScan was used to predict targets of miR-340. A dual luciferase reporter assay was performed to confirm a potential direct interaction between miR-340-5p and ZNF503. Results The expression level of miR-340-5p was frequently found to be lower in NSCLC tissues than in matched pericarcinomatous normal tissues. Overexpression of miR-340-5p significantly inhibited the proliferation and invasion NCI-H1650 (a NSCLC cell line), while inhibition of miR-340-5p stimulated cell growth. Using TargetScan, we predicted that ZNF503 could be a target of miR-340-5p. Further mechanistic studies demonstrated that the forced expression of ZNF503 could partially abrogate the miR-340-5p-mediated decrease in NCI-H1650 cell viability and invasion, suggesting that miR-340-5p suppressed cell growth and invasion in a ZNF503-dependent manner. Conclusion Our findings indicate that miR-340-5p inhibits NCI-H1650 cell proliferation and invasion by directly targeting ZNF503 and that miR-340-5p can serve as a potential therapeutic target for treating NSCLC. Electronic supplementary material The online version of this article (10.1186/s11658-019-0161-1) contains supplementary material, which is available to authorized users.

The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1. Then, the complexes were used to transfect three Siberian sturgeon cell lines derived from the heart, head kidney, and gonad. Cytotoxicity and transfection efficiency were measured via flow cytometry after propidium iodide staining. No significant cytotoxicity was observed at the optimal treatment conditions in all cases, with the exception of Lipofectamine 2000-treated gonad-derived cells. Although the transfection efficiencies in A. baerii cells were generally low, X-tremeGENE HP DNA Transfection Reagent showed the highest transfection efficiency at ratios of 2:1 or 4:1, depending on the cell type. Hence, X-tremeGENE HP DNA Transfection Reagent can be used to effectively transfer foreign genes into three A. baerii cell lines.

Objectives Heat treatment as a physical method could increase the cellular uptake of nucleic acids. In this study, the effects of heat shock were evaluated to enhance the transfection efficiency of three plasmid DNAs into HeLa and TC-1 cancerous, and HEK-293 T and Vero non-cancerous cell lines using lipofectamine 2000 reagent. Methods Two methods of cell- and DNA-based heat treatment were used. Heating DNA solution was performed at 94 °C for 5, 10 and 15 min, and also 72 °C for 30, 60 and 120 min, individually. Moreover, heating the cells was done by incubation at 42 °C for 2 h in different times such as before, during and after DNA transfection. Results Our data showed that the conformation of plasmid DNAs was changed at different temperatures with increasing time. The heat-treated plasmid DNAs (94 °C for 10 min or 72 °C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs (p

Purpose: To study the effect of miR-23b on the expression of the target gene of acetaldehyde dehydrogenase 1A1 (ALDH1A1), and cisplatin (CDDP) susceptibility of cervical carcinoma stem cells. Methods: Human cervical cancer cell line Hela cells were cultured in vitro, and miR-23b mimic and negative control were transfected into the cells using lipofectamine method. The growth of the two groups of cells was determined using growth curve method, and their proliferation measured using plate clone formation. The influence of treatments on the sensitivity of the cells to CDDP was assayed using MTT method. The mRNA expression of ALDH1A1 in Hela cells was assayed using real-time quantitative polymerase hain reation (PCR), while its protein expression was assayed by Western blot. Results: The levels of expressions of ALDH1A1 protein and mRNA in the miR-23b overexpression group were significantly lower than those in the control group (p < 0.05). The sensitivities of Hela cells to CDDP in the ALDH1A1 inhibition group and the control group were dose-dependent to some extent, but cell inhibition in ALDH1A1 inhibition group markedly increased, relative to control when the CDDP dose was 0.1 ppc (p < 0.01). Conclusion: Up-regulating the expression of miR-23b significantly inhibits the growth and proliferation of cervical cancer cells, and increases their sensitivity to CDDP via down-regulation of the expression of the target gene for ALDH1A1. Therefore, during cervical carcinoma treatment, increasing the level of miR-23b may produce a chemotherapeutic effect. Keywords: MiR-23b, Acetaldehyde dehydrogenase 1A1, Cervical cancer, Cisplatin, Sensitivity

BACKGROUND MicroRNAs (miRNAs) are novel biomarkers that are important in tumorigenesis and cancer treatment resistance. miR-451 is expressed in human papillary thyroid carcinoma (PTC) tissues and is associated with tumor progression. This study investigated the molecular mechanism associated with the effects of miR-451 on B-CPAP human PTC cells in vitro. MATERIAL AND METHODS Binding of miRNAs to the 3' untranslated region (3'UTR) of messenger RNA (mRNA) was determined with a luciferase reporter assay. miRNAs and plasmids were transfected into human PTC B-CPAP cells with Lipofectamine 2000 Transfection Reagent. Cell viability was tested with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. The levels of miRNAs and mRNA were determined with quantitative polymerase chain reaction and protein levels were analyzed with immunoblotting. RESULTS miR-451 bound to wild-type but not mutant 3'-UTR of activating transcription factor 2 (ATF2). MiR-451 mimics inhibited the growth of B-CPAP cells and reduced mRNA and protein levels in ATF2, whereas miR-451 inhibitors promoted the growth of B-CPAP cells and increased mRNA and protein levels in ATF2. CONCLUSIONS miR-451 directly bound to the 3'UTR of ATF2, decreased mRNA and protein levels in ATF2, and inhibited growth of B-CPAP cells. Our findings suggest that miR-451 may be a potential therapeutic target for PTC.

Background One of the major challenges in gene therapy is producing gene carriers that possess high transfection efficiency and low cytotoxicity (1). To achieve this purpose, crystal nanocellulose (CNC) -based nanoparticles grafted with polyethylenimine (PEI) have been developed as an alternative to traditional viral vectors to eliminate potential toxicity and immunogenicity. Methods In this study, CNC-PEI10kDa (CNCP) nanoparticles were synthetized and their transfection efficiency was evaluated and compared with linear cationic PEI10kDa (PEI) polymer in HEK293T (HEK) cells. Synthetized nanoparticles were characterized with AFM, FTIR, DLS, and gel retardation assays. In-vitro gene delivery efficiency by nano-complexes and their effects on cell viability were determined with fluorescent microscopy and flow cytometry. Results Prepared CNC was oxidized with sodium periodate and its surface cationized with linear PEI. The new CNCP nano-complex showed different transfection efficiencies at different nanoparticle/plasmid ratios, which were greater than those of PEI polymer. CNPC and Lipofectamine were similar in their transfection efficiencies and effect on cell viability after transfection. Conclusion CNCP nanoparticles are appropriate candidates for gene delivery. This result highlights CNC as an attractive biomaterial and demonstrates how its different cationized forms may be applied in designing gene delivery systems.

Polyethylenimines (PEIs) have been shown as efficient gene delivery vectors due to their unique properties, however, toxicity as well as non-specific interactions with the tissues/cells because of high charge density have hampered their use in clinical applications. To counter these concerns, here, we have prepared disachharide-PEI organic nanoparticles by mixing PEIs with non-reducing disaccharides, i.e. trehalose (TPONs) and sucrose (SPONs), under mild conditions. The fabricated nanoparticles were complexed with pDNA and size of these complexes was found in the range of ~130–162 nm with zeta potential ~ +8–25 mV. Further evaluation of these nanoparticles revealed that substitution of disaccharides on PEIs successfully augmented cell viability. Transfection efficiency exhibited by these complexes was significantly higher than the unmodified polymer and the standard, Lipofectamine, complexes. Fabrication of organic nanoparticles did not alter the buffering capacity considerably which was found to be instrumental during endosomal escape of the complexes. Among both the series of nanoparticles, trehalose-PEI organic nanoparticles (TPONs) exhibited greater pDNA transportation potential than sucrose-PEI organic nanoparticles (SPONs) which was also established by flow cytometric data, wherein percent cells expressing GFP was higher in case of TP/pDNA complexes as compared to SP/pDNA complexes. Interestingly, TPONs also showed promising anticancer activity on cancer cell lines i.e. Mg63, MCF-7 and HepG2. Overall, the results advocate promising potential of disaccharide-PEI organic nanoparticles as efficient gene delivery agents which can be used effectively in future gene therapy applications along with anti-cancer competence of TPONs.

Objective. Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods. BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results. miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions. miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

Funding: Work on plant-parasitic nematodes at the University of Cambridge is supported by DEFRA licence 125034/359149/3, and was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/R011311/1, BB/N021908/1, and BB/S006397/1, a Synthego Genome Engineer grant, and a Genewiz grant. A.A. was supported by a Wellcome/Newton trust Institutional Strategic Support Fund grant and a UKRI Future Leaders Fellowship MR/S033769/1. C.J.L. was supported by BBSRC grant BB/N016866/1. J.J. receives funding from the Scottish Government Rural and Environmental Science and Analytical Services division. T.R.M. and T.J.B. were supported by grants from the Iowa Soybean Association and by State of Iowa and Hatch funds. A.P.S. and S.A. were supported by grants RPG-2016-089 and BB/L019884/1. Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security. Publisher PDF

Cells from different origins behave differently regarding the incorporation of exogenous DNA and formation of transgenic cells. Milk production of recombinant antibody may benefit from efficient transfection protocols to produce transgenic animals. In this context, the objective of this study was to verify the transfection potential of bovine mesenchymal stem cells from Wharton's jelly (MSC-WJ) and adipose tissue (MSC-AT), comparing co-transfection protocols with vectors pBC1-anti-CD3 and pEF-NEO-GFP, using transfection reagents Lipofectamine LTX with Plus Reagent or Xfect. Skin fibroblasts (FIB) were used as the control group. Forty-eight hours after transfection, neomycin was added and cells cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than fibroblasts, for both the Xfect (20.057 ± 1.620,7 and 10.601 ± 702,86, respectively, p

Introduction The biological role of specific genes can be investigated using gene silencing by cell transfection with small interfering RNA (siRNA), which leads to degradation of the corresponding mRNA and reduced target protein expression. The study of biological functions in modified cell cultures often requires a long-lasting knockdown (KD) of gene expression, which can be obtained by viral delivery of short hairpin RNA (shRNA) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 systems. However, these methods are laborious and require high level of laboratory safety. Hematopoietic cells, typically grown in suspension, are known to be difficult to transfect. Previous efforts to transfect lymphocytes using liposomes (H Guven et al, ExpHem2005) showed limited transfection efficiencies of only 10-30%. Transfection by electroporation leads to decreased viability, and therefore poses a risk of unintentional selection of cell subsets. Transfection of leukemia cells with siRNAs using Accell siRNA Delivery Media showed KD efficiencies of 56-65% and a viability of 57-95% (H Larsen et al,ExpHem2011). However, this was only investigated 24-72h post-transfection. By whole transcriptome sequencing, we previously identified a number of candidate genes, includingLILRA4, PTPRJ and SYNE2, which were significantly upregulated in mantle cell lymphoma (MCL) cells compared to B cells from healthy donors (MH Hansen et al, ExpHem 2020). To investigate the biological function of these genes, an efficient and stable KD was required. Aim The aim of this study was to identify the most effective non-viral method for siRNA-mediated knockdown in MCL cells. Methods The MCL cell lines Mino, Jeko-1 and Granta 519, were transfected using three different methodologies; Lipofectamine 2000 (Invitrogen), Electroporation with Amaxa Nucleofector (Lonza), and Accell siRNA Delivery Media (Dharmacon). For Accell delivery a pool of 4 siRNAs targeting each gene was used (SMARTpool siRNA, Dharmacon). For the others, 2 pre-designed siRNAs (Ambion) targeting each gene were used. For all, a positive control siRNA targetingGAPDHwas included and different conditions tested e.g. siRNA concentration, incubation time and addition of serum. KD efficiency was evaluated by qPCR usingABLandGUSreference genes. Viability of cells was measured either by flow cytometry staining with a live/dead marker or by trypan blue. Optimization of the Accell protocol was performed in Granta 519 cells transfected with SMARTpool siRNAs targeting eitherSYNE2,LILRA4,orGAPDH. KD efficiency was evaluated with qPCR and viability was counted with trypan blue. Results Using Lipofectamine, at 24h a non-reproducible and non-efficient KD ( Conclusion This study propose that transfection with SMARTpool siRNAs in Accell Delivery Medium with addition of 10% FBS 48h post transfection is an applicable procedure for siRNA-mediated KD in MCL suspension cell lines. This protocol permits efficient, non-viral, and stable KD across different MCL cell lines without affecting the cells' ability to proliferate and survive, and is therefore suitable for investigating biological processes. Disclosures No relevant conflicts of interest to declare.

The present study compared the effects of ultrasonic irradiation and SonoVue microbubbles (US) or Lipofectamine 3000 on the transfection of small interfering RNA for PRR11 (siPRR11) and Proline-rich protein 11 (PRR11) overexpression plasmid into breast cancer cells. SiPRR11 and PRR11 overexpression plasmid were transfected into breast cancer MCF7 cells mediated by US and Lipofectamine 3000. PRR11 expressions in breast cancer and normal tissues were determined using Gene Expression Profiling Interactive Analysis (GEPIA). The viability, proliferation, migration, invasion and apoptosis of breast cancer cells were respectively measured by MTT assay, clone formation assay, scratch wound-healing assay, Transwell assay and flow cytometry. PRR11 and epithelial-to-mesenchymal transition (EMT)-related and apoptosis-related (B-cell lymphoma 2, Bcl-2; Bcl-2-associated protein X, Bax) proteins’ expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as appropriate. As ultrasonic intensity increased, the viability of MCF7 cells was decreased. Results from GEPIA suggested that PRR11 was up-regulated in breast cancer. Silencing PRR11 mediated by US showed a higher efficiency than by Lipofectamine 3000. SiPRR11 transfected by Lipofectamine 3000 suppressed cells growth and metastasis, while promoted cell apoptosis. Moreover, E-cadherin (E-cad) and Bax expressions were high but N-cadherin (N-cad), Snail and Bcl-2 expressions were low. However, overexpressed PRR11 caused the opposite effects. More importantly, transfection of siPRR11 and PRR11 overexpression plasmid using US had a higher efficacy than using Lipofectamine 3000. US transfection of PRR11 siRNA showed better effects on inhibiting breast cancer progression. The current findings contribute to a novel treatment for breast cancer.

Self-amplifying(sa) RNA molecules – “replicons” – derived from the genomes of positive-sense RNA are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a non-monotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-sa RNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a “sweet spot” at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of lipofectamine, the replicons were mixed with a “carrier” RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as one tenth of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements.

In this study, indole-3-butanoic acid (IBA), a biologically and environmentally safe entity, has been grafted onto low and high molecular weight (1.8 and 25 kDa) polyethylenimines (PEI) mainly through primary amines to obtain amphiphilic indole-3-butanoyl-polyethylenimines (IBPs). Two series of IBPs (IBP1.8 and IBP25) were prepared which, on self-assembly in aqueous medium, yielded multifunctional nanomicellar structures (IBP1.8 and IBP25) capable of transporting genetic material in vitro and exhibiting other biological activities. Physicochemical characterization showed the size of IBP1.8 and IBP25 nanostructures in the range of ~332–234 nm and ~283–166 nm, respectively, with zeta potential varying from ~+29–17 mV and ~+37–25 mV. DNA release assay demonstrated higher release of plasmid DNA from IBP nanostructures as compared to native PEIs. Cytotoxicity showed a decreasing pattern with increasing degree of grafting of IBA onto PEIs making these nanostructures non-toxic. pDNA complexes of these nanostructures (both IBPs1.8 and IBPs25) displayed considerably higher transfection efficiency, however, IBP1.8/pDNA complexes performed much better (~7–9 folds) as compared to native PEI/pDNA and Lipofectamine/pDNA complexes on mammalian cells. CLSM analysis revealed that these complexes entered nucleus in sufficient amounts suggesting higher uptake and efficient internalization of the complexes. Besides, these supramolecular nanostructures not only exhibited excellent antimicrobial potential (MIC ~49–100 µg/ml) against clinical as well as resistant pathogenic strains but also found to possess antioxidant property. Overall, the projected low molecular weight PEI-based vectors could serve as more effective multifunctional nanomaterials having promising potential for future gene therapy applications with capability to provide protection against other bacterial infections.

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.

Cellulose-based nanoparticles were prepared from oppositely charged carboxymethyl cellulose (CMC) and quaternized cellulose (QC) in an aqueous medium. The DNA complexing capacity of CMC–QC nanoparticles and the transfection efficiency of the DNA-loaded nanoparticles in COS-7 cells were first investigated using pEGFP-N1—a plasmid DNA encoding the enhanced green fluorescent protein (EGFP)—as a model, and then with a candidate DNA vaccine, pMASIA-tPAs-tE2.2, that has been developed against infection with bovine viral diarrhea virus (BVDV). The results revealed that CMC–QC nanoparticles could bind DNA efficiently, and the optimized DNA-loaded nanoparticles mediated very effective transfection in COS-7 cells, which was comparable to that achieved with Lipofectamine 2000. The novel CMC–QC nanoparticles show promise as a delivery system for DNA vaccines.

BACKGROUND The present study explored the role and mechanism of microRNA-874-3p (miR-874-3p) in the migration of the osteosarcoma cell line, U-2 OS. METHODS The expression profile of osteosarcoma (OS) microRNA (GSE65071) datasets was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) to identify differentially expressed miRNAs in OS and its biological functions. A quantitative reverse transcription-polymerase chain reaction was performed to detect the expression of miR-874-3p and its target gene regulator of G protein 4 (RGS4) in human osteosarcoma cells U-2 OS and normal osteoblast hFOB1.19. Plasmid overexpression miR-874-3p and pcDNA-RGS4 were transfected into U-2 OS using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA). Cell migration was measured using Transwell migration assays. Bioinformatic analysis and luciferase reporter assay were conducted to search for the target gene of miR-874-3p. RESULTS In total, 167 differentially expressed miRNAs were detected after the analysis of GSE65071; of which 78 were up-regulated genes and 89 were down-regulated. miR-874-3p was down-regulated and selected for further analysis. The expression level of miR-874-3p in U-2 OS cells was significantly decreased compared to the hFOB1.19 cell line (p < 0.05). Overexpression of miR-874-3p significantly inhibited the proliferation and migration of U-2 OS cells and overexpression of RGS4 reversed the inhibitory effect of miR-874-3p on U-2 OS cells. Through luciferase report analyses and bioinformatic analysis, RGS4 may be the candidate target gene of miR-874-3p. CONCLUSIONS In conclusion, overexpression of miR-874-3p suppressed OS cell proliferation and migration. Thus, miR-874-3p might present a therapeutic agent for the treatment of OS.

Development of non-viral gene vectors which can efficiently and safely transfect plasmid DNA and siRNA into cells is of great importance for gene therapy. Despite lots of efforts spent, it is still imperative to develop suitable gene vectors with better transfection efficiency and low cytotoxicity. To this end, we successfully designed, synthesized and screened a library of 120 polymers (via nucleophilic substitution reaction between dihalides and amines). With cell-based transfection screening assays, 120 polymers were tested to evaluate their transfection efficiency of transporting DNA and siRNA into cells. Our results indicated that hydrophobic modification could greatly enhance cationic polymers’ transfection efficiency, and polymers with long linkers usually showed better transfection performance, especially for polymers with the linker of 1, 12-dibromododecane (L3 linker). Besides, polyalkylamines exhibited better transfection efficiency with the polymer particle size around 200 nm and the zeta potential in the range of + 40 mV to +50 mV. Interestingly, polymer particles made from N15HL3 not only exhibited better DNA transfection efficiency in HEK 293T cells but also showed higher siRNA transfection efficiency in U87 Luc-GFP cells together with low cell toxicity than Lipofectamine 2000 (one of commercial transfection reagents). Therefore, it is hoped that our study here not only provides promising gene vector candidates for further evaluation in gene therapy, but also provides valuable insights for better understanding of the relationship between the chemical structures and gene transfection efficiency to rationally design better non-viral gene vectors for gene therapy in the future.

The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra-and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy. �� 2020 by the authors.

Low-intensity pulsed ultrasound (LIPUS), a special type of ultrasonic stimulation, is attracting a lot of attention for both clinical and scientific research. In this paper, we report a concept of a new method using magnetic nanoparticles (MNPs) for LIPUS-assisted gene delivery. The MNPs are iron oxide superparamagnetic nanoparticles, coated with polyethyleneimine (PEI), which introduces a high positive surface charge, favorable for the binding of genetic material. Due to the paramagnetic properties of the MNPs, the application of an external magnetic field increases transfection efficiency; meanwhile, LIPUS stimulation enhances cell permeability. We found out that stimulation at the intensity of 30 mW/cm2 for 10 minutes yields optimal results with a minimal adverse effect on the cells. Combining the effect of the external magnetic field and LIPUS, the genetic material (GFP or Cherry Red plasmid in our case) can enter the cells. The flow cytometry results showed that by using just a magnetic field to direct the genetic material, the transfection efficiency of HEK 293 cells that were treated with our MNPs was 56.1%. Coupled with LIPUS stimulation, it increased to 61.5% or 19% higher than the positive control (Lipofectamine 2000). In addition, compared with the positive control, our method showed less toxicity. Cell viability after transfection was 63.61%, 19% higher than with the standard transfection technique. In conclusion, we designed a new gene-delivery technique that is affordable, targeted, shows low-toxicity, yet high transfection efficiency, compared to other conventional approaches.The Graphical Abstract

Background: β-glucan from Lentinus edodes (LNT) is a plant-derived medicinal fungus possessing significant bioactivities on anti-tumor. Both hypoxia-induced factor-1α (HIF)-1α and Nur77 have been shown to be involved in the development of breast cancer. However, there is yet no proof of Nur77/HIF-1α involvement in the process of LNT-mediated tumor-inhibition effect. Methods: Immunohistochemistry, immunofluorescence and Hematoxylin–Eosin staining were used to investigate tumor growth and metastasis in MMTV-PyMT transgenic mice. Proliferation and metastasis-associated molecules were determined by Western blotting and reverse transcription-quantitative PCR. Hypoxic cellular model was established under the exposure of CoCl2. Small interference RNA was transfected using Lipofectamine reagent. The ubiquitin proteasome pathway was blunted by adding the proteasome inhibitor MG132. Results: LNT inhibited the growth of breast tumors and the development of lung metastases from breast cancer, accompanied by a decreased expression of HIF-1α in the tumor tissues. In in vitro experiments, hypoxia induced the expression of HIF-1α and Nur77 in breast cancer cells, while LNT addition down-regulated HIF-1α expression in an oxygen-free environment, and this process was in a manner of Nur77 dependent. Mechanistically, LNT evoked the down-regulation of HIF-1α involved the Nur77-mediated ubiquitin proteasome pathway. A strong positive correlation between Nur77 and HIF-1α expression in human breast cancer specimens was also confirmed. Conclusion: Therefore, LNT appears to inhibit the progression of breast cancer partly through the Nur77/HIF-1α signaling axis. The findings of the present study may provide a theoretical basis for targeting HIFs in the treatment of breast cancer.

Gene transfection is important in biotechnology and is used to modify cells intrinsically. It can be conducted in cell suspension or after cell adhesion, where the efficiency is dependent on many factors such as the type of nanocarrier used and cell division processes. Anchor-dependent cells are sensitive to the substrate they are attached to and adapt their behavior accordingly, including plasmid trafficking during gene transfection. Previously, it was shown in our group that the cytoskeleton is an essential factor in influencing gene transfection in skeletal myoblasts using nanogrooves as a substrate. In this study, the effect of the cytoskeleton on gene transfection efficiency of skeletal myoblasts was studied using various nanopillars and nanocarriers. Nanopillars with different diameters (200-1000 nm) and depths (200 or 400 nm) were fabricated using colloidal self-assembly and reactive ion etching. All surfaces were treated with oxygen plasma or polydopamine (PD) to further control cell morphology. Plasmid DNA was delivered into cells using jetPRIME or Lipofectamine 3000 nanocarriers. After screening hundreds of images, two distinguishable F-actin distributions were found, i.e., cells with or without a perinuclear actin cap (pnAC). Cells attached to nanopillars, especially the deep pillars, had a smaller spreading area, shorter F-actin, more 3D-like cell nuclei, and a lower percentage of pnAC, which lead to a higher gene transfection efficiency using jetPRIME. On the other hand, cells attached to the shallow nanopillars or flat surfaces had a larger spreading area, longer F-actin, more 2D-like cell nuclei, and a higher percentage of pnAC that facilitates gene transfection using Lipofectamine. The effects of cell density, cytoskeleton (cytoD), and focal adhesions (RGD) on gene transfection were also studied, and the results were consistent with our hypothesis that F-actin distribution is one of the critical factors in gene transfection. In conclusion, pnAC plays a vital role in the intracellular trafficking of nanocarrier/plasmid complexes and this study provides new insights into gene transfection in anchor-dependent cells. STATEMENT OF SIGNIFICANCE: This study provides a new perspective in gene transfection using attached cells where perinuclear actin cap (pnAC) is an essential factor involved in transfection efficiency. A series of nanopillars were used to harness cell and cytoskeleton morphology. Two distinguishable cytoskeletal structures were found including cells with or without pnAC. 2D-like cells with pnAC facilitate gene delivery using liposome-based nanocarriers, while 3D-like cells without pnAC benefit gene delivery using cationic polymer-based nanocarriers. This study reveals the importance of the cytoskeleton during gene transfection that is beneficial in tissue transfection.

Anti-influenza drugs and vaccines have a limited effect due to the high mutation rate of virus genome. The direct impact on the conservative virus genome regions should significantly improve therapeutic effectiveness. The RNA interference mechanism (RNAi) is one of the modern approaches used to solve this problem. In this work, we have investigated the antiviral activity of small interfering RNA (siRNA) against the influenza A/PR/8/34 (H1N1), targeting conserved regions of NP and PA. Polycations were used for intracellular siRNA delivery: chitosan's derivatives (methylglycol and quaternized chitosan), polyethyleneimine, lipofectamine, and hybrid organic/non-organic microcapsules. A comparative study of these delivery systems with fluorescent labeled siRNA was conducted. The antiviral activity of three small interfering RNAs targeting the NP (NP-717, NP-1496) and PA (PA-1630) influenza A viruses genes was demonstrated, depending on the chosen carrier. The most effective intracellular delivery and antiviral activity were observed for hybrid microcapsules.Механизм РНК-интерференции открывает широкие возможности при создании новых препаратов для лечения гриппа. Он позволяет точечно воздействовать на консервативные участки вирусных генов и блокировать их экспрессию. Проведена сравнительная оценка различных носителей для внутриклеточной доставки малых интерферирующих РНК: метилгликольхитозана, кватернизованного хитозана, полиэтиленимина и гибридных микрокапсул на основе полиаргинина с неорганическими компонентами. Кроме того, оценивали противовирусную активность трех малых интерферирующих РНК, направленных на гены NP (NP-717, NP-1496) и PA (PA-1630) вирусов гриппа А, в зависимости от выбранного носителя. По результатам исследования наиболее эффективные внутриклеточная доставка и противовирусная активность были показаны для гибридных микрокапсул.