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Transfection of mammalian and human cell lines in medical research both are key technologies in molecular biology and genetic engineering. A vast variety of techniques to facilitate transfection exists including different chemical and nanoparticle-based agents as mediators of nucleic acid uptake, with nanoparticles composed of the lipids DOSPA/DOPE belonging to the established type of agents. We show that inverse-nanoemulsion-derived protein nanohydrogels (NanoTrans-gels), prepared by a simple synthesis protocol, are suited to transfect two model cancer cell lines (MCF7 and A549) with high efficiency. The transfection efficiency was analyzed in comparison to the DOSPA/DOPE-dependent protocols as a reference method. Since nanogel-based transfection outperformed the Lipofectamine-dependent technique in our experiments, we believe that the NanoTrans-gels loaded with plasmid DNA may open new avenues for simple and efficient transfection for humans and probably also other mammalian cell lines and may develop into a general tool for standard transfection procedures in cell biology laboratories. [ABSTRACT FROM AUTHOR]

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida–free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency. [ABSTRACT FROM AUTHOR]

Transfection of mammalian and human cell lines in medical research both are key technologies in molecular biology and genetic engineering. A vast variety of techniques to facilitate transfection exists including different chemical and nanoparticle-based agents as mediators of nucleic acid uptake, with nanoparticles composed of the lipids DOSPA/DOPE belonging to the established type of agents. We show that inverse-nanoemulsion-derived protein nanohydrogels (NanoTrans-gels), prepared by a simple synthesis protocol, are suited to transfect two model cancer cell lines (MCF7 and A549) with high efficiency. The transfection efficiency was analyzed in comparison to the DOSPA/DOPE-dependent protocols as a reference method. Since nanogel-based transfection outperformed the Lipofectamine-dependent technique in our experiments, we believe that the NanoTrans-gels loaded with plasmid DNA may open new avenues for simple and efficient transfection for humans and probably also other mammalian cell lines and may develop into a general tool for standard transfection procedures in cell biology laboratories.

Purpose: Non-viral transfection approaches are extensively used in cancer therapy. The future of cancer therapy lies on targeted and efficient drug/gene delivery. The aim of this study was to determine the transfection yields of two commercially available transfection reagents (i.e. Lipofectamine 2000, as a cationic lipid and PAMAM G5, as a cationic dendrimer) in two breast cell lines: cancerous cells (T47D) and non-cancerous ones (MCF-10A). Methods: We investigated the efficiencies of Lipofectamine 2000 and PAMAM G5 for transfection/delivery of a labeled short RNA into T47D and MCF-10A. In addition to microscopic assessments, the cellular uptakes of the complexes (fluorescein tagged-scrambled RNA with Lipofectamine or PAMAM dendrimer) were quantified by flow cytometry. Furthermore, the safety of the mentioned reagents was assessed by measuring cell necrosis through the cellular PI uptake. Results: Our results showed significantly better efficiencies of Lipofectamine compared to PAMAM dendrimer for short RNA transfection in both cell types. On the other hand, MCF-10A resisted more than T47D to the toxicity of higher concentrations of the transfection reagents. Conclusion: Altogether, our research demonstrated a route for comprehensive epigenetic modification of cancer cells and depicted an approach to efficient drug delivery, which eventually improves both short RNA-based biopharmaceutical industry and non-viral strategies in epigenetic therapy.

Nucleic acid nanoparticles (NANPs) require a carrier to allow for their intracellular delivery to immune cells. Cytokine production, specifically type I and III interferons, allows for reliable monitoring of the carrier effect on NANP immunostimulation. Recent studies have shown that changes in the delivery platform (e.g., lipid-based carriers vs. dendrimers) can alter NANPs' immunorecognition and downstream cytokine production in various immune cell populations. Herein, we used flow cytometry and measured cytokine induction to show how compositional variations in commercially available lipofectamine carriers impact the immunostimulatory properties of NANPs with different architectural characteristics. [ABSTRACT FROM AUTHOR]

Purpose: Non-viral transfection approaches are extensively used in cancer therapy. The future of cancer therapy lies on targeted and efficient drug/gene delivery. The aim of this study was to determine the transfection yields of two commercially available transfection reagents (i.e. Lipofectamine 2000, as a cationic lipid and PAMAM G5, as a cationic dendrimer) in two breast cell lines: cancerous cells (T47D) and non-cancerous ones (MCF-10A). Methods: We investigated the efficiencies of Lipofectamine 2000 and PAMAM G5 for transfection/delivery of a labeled short RNA into T47D and MCF-10A. In addition to microscopic assessments, the cellular uptakes of the complexes (fluorescein tagged-scrambled RNA with Lipofectamine or PAMAM dendrimer) were quantified by flow cytometry. Furthermore, the safety of the mentioned reagents was assessed by measuring cell necrosis through the cellular PI uptake. Results: Our results showed significantly better efficiencies of Lipofectamine compared to PAMAM dendrimer for short RNA transfection in both cell types. On the other hand, MCF-10A resisted more than T47D to the toxicity of higher concentrations of the transfection reagents. Conclusion: Altogether, our research demonstrated a route for comprehensive epigenetic modification of cancer cells and depicted an approach to efficient drug delivery, which eventually improves both short RNA-based biopharmaceutical industry and non-viral strategies in epigenetic therapy. [ABSTRACT FROM AUTHOR]

To assess in vitro and in vivo tracking of iron oxide labeled stem cells transfected by lipofectamine using magnetic resonance imaging (MRI), rat dental pulp stem cells (DPSCs) were characterized, labeled with iron oxide nanoparticles, and then transfected with lipofectamine to facilitate the internalization of these nanoparticles. Cell proliferation, viability, differentiation, and apoptosis were investigated. Prussian blue staining and MRI were used to trace transfected labeled cells. DPSCs were a morphologically spindle shape, adherent to culture plates, and positive for adipogenic and osteogenic inductions. They expressed CD73 and CD90 markers and lacked CD34 and CD45. Iron oxide labeling and transfection with lipofectamine in DPSCs had no toxic impact on viability, proliferation, and differentiation, and did not induce any apoptosis. In vitro and in vivo internalization of iron oxide nanoparticles within DPSCs were confirmed by Prussian blue staining and MRI tracking. Prussian blue staining and MRI tracking in the absence of any toxic effects on cell viability, proliferation, differentiation, and apoptosis were safe and accurate to track DPSCs labeled with iron oxide and transfected with lipofectamine. MRI can be a useful imaging modality when treatment outcome is targeted. [ABSTRACT FROM AUTHOR]

Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A22/IRQ/24/64) into the tongue epithelium. All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals. [ABSTRACT FROM AUTHOR]

Cell-based microarrays are valuable tools for analyzing cellular functions. However, a significant limitation of conventional microarrays is their inapplicability to non-adherent cells. In this study, we investigated the potential of the 'Cell Dome' (diameter: 1 mm, height: approximately 300 µm) with a 90 µm-thick hydrogel shell as a gene transfection array for non-adherent cells in suspension. The human lymphoma cell line (K562 cells), used as a model for non-adherent cells, was transfected more efficiently by Lipofectamine/pDNA complexes on a composite hydrogel made of polyvinyl alcohol derivative (PVA-Ph) and chitosan derivative (chitosan-Ph) than hydrogels composed of an alginate derivative or PVA-Ph alone. Moreover, no significant adverse effects on the viability and proliferation of the enclosed cells were observed for Cell Dome with a PVA-Ph/chitosan-Ph composite hydrogel shell. Lipofectamine/pDNA complexes released from the bottom of Cell Domes could transfect the enclosed cells without leaking or contaminating adjacent Cell Domes. These results demonstrate the potential of Cell Domes with an appropriate hydrogel shell as transfection arrays for non-adherent cells in suspension, thereby expanding the range of applications of cell-based array technologies. This novel Cell-Dome transfection array would be a valuable tool for analyzing the cellular function of non-adherent cells in suspension and showcases the potential for providing important biomedical insights for future research and developments. [Display omitted] • Cell Dome enables effective transfection of non-adherent cells in suspension. • PVA-Ph and chitosan-Ph composite hydrogels formed transfection-efficient Cell Dome shells. • Lipofectamine/pDNA-based Cell Dome system allows transfection of enclosed non-adherent cells in suspension. • No transfection leakage or contamination in adjacent Cell Domes ensures reliability. [ABSTRACT FROM AUTHOR]

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.

Nucleic acid nanoparticles (NANPs) require a carrier to allow for their intracellular delivery to immune cells. Cytokine production, specifically type I and III interferons, allows for reliable monitoring of the carrier effect on NANP immunostimulation. Recent studies have shown that changes in the delivery platform (e.g., lipid-based carriers vs. dendrimers) can alter NANPs’ immunorecognition and downstream cytokine production in various immune cell populations. Herein, we used flow cytometry and measured cytokine induction to show how compositional variations in commercially available lipofectamine carriers impact the immunostimulatory properties of NANPs with different architectural characteristics.

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 hIFNβ1apcDNA3.1 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. [ABSTRACT FROM AUTHOR]

Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semirandom patterns, and their transduction efficiency varies between patients. In this study, several nonviral gene delivery vehicles, promoters, and additional variables were compared to optimize nonviral transgene delivery and expression in both Jurkat and primary T cells. Transfection of Jurkat cells was maximized to a high efficiency (63.0% ± 10.9% EGFP+ cells) by transfecting cells with Lipofectamine LTX in X‐VIVO 15 media. However, the same method yielded a much lower transfection efficiency in primary T cells (8.1% ± 0.8% EGFP+). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans detected via messenger RNA‐sequencing. Pyrin and HIN (PYHIN) DNA sensors (e.g., AIM2 and IFI16) that can induce apoptosis or repress transcription after binding cytoplasmic DNA were also detected at high levels in primary T cells. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake or DNA sensing in the cytoplasm. Both of these factors should be considered in the development of future viral and nonviral T cell gene delivery methods. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and Objectives: Nonviral carriers including those based on synthetic cationic lipids, offer several advantages over the viral counterparts. These carriers are able to form complexes with nucleic acids and deliver genes into the cells via the cellular endocytosis pathway, without significant toxicity. The level of transgenes expression depends on some experimental variables including cell type and density, Lipofectamine and DNA concentrations and Lipofectamine-DNA complexing time. The main objective of this study was to optimize transfection of SW480 colon cancer cells with Lipofectamine 2000. Methods: In this study, SW480 cells were transfected with plasmid containing green fluorescent protein reporter gene using Lipofectamine 2000. Green fluorescent protein expression was studied under a reverse fluorescence microscope and the results were analyzed with the ImageJ software. Effect of different quantities of plasmid DNA and different Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Results: The optimal volume of Lipofectamine and quantity of plasmid was 2 µl and 1µg, respectively, which showed 59% efficiency for the transfection of SW480 cells at 24 hours post-transfection. Conclusion: This study shows that Lipofectamine 2000 is an efficient reagent for the delivery of genes into SW480 cells. According to the results, the quantity of DNA per transfection and reagent concentrations are essential factors for a successful transfection. Keywords: Optimization; pEGFP-NI; Lipofectamine; SW480.

Lipofectamine is a commonly used transfection reagent in molecular biology. It's a lipid-based molecule that helps introduce foreign genetic material (like DNA or RNA) into cells. This study explored the potential of Lipofectamine to kill breast cancer cells (MCF7 line) using the MTT assay. The miRNA mimics, inhibitors, and negative control (NC) were synthesized and labeled with Stealth/siRNA Transfection Protocol Lipofectamine 2000 ThermoFisher. The study employed both miRNA-199a-5p mimic and inhibitor groups. While the mimic augmented miRNA-199a-5p function, the inhibitor suppressed it. Statistical significance was determined by p-values below 0.05. Both the mimic (p=0.0084) and inhibitor (p=0.0113) groups produced statistically significant results. In conclusion, Lipofectamine demonstrated efficacy in transferring miRNA into MCF-7 cells while causing minimal cellular damage. Peak miRNA expression was observed 72 hours after transfection. Modulation of miRNA-199a-5p levels substantially affected the survival of breast cancer cells. [ABSTRACT FROM AUTHOR]

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

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.

AIM: To investigate the expression of connective tissue growth factor(CTGF)and α-SMA in human lens epithelium cell(HLEC)line B3 after transfection by liposome-coated siRNA targeting CTGF. METHODS: HLECs were transfected with small interfering RNA(siRNA)targeting CTGF, labeled with 5'- fluorescein isothiocyanate(5'-FITC)and coated with lipofectamine. The transfection ratio was evaluated via fluorescence intensity. Cell counting kit-8(CCK-8)assay was performed to assess cytoviability of both non-transfected and transfected HLECs. Quantitative RT- PCR, cell immunochemistry and Western blot analysis were conducted to detect the expression changes of CTGF and α-SMA after transfection. RESULTS: A highly effective transfection ratio was observed in siRNA co-transfected with lipofectamine. The transfection ratio reached 95% at 24h. The proliferation of HLECs was inhibited by siRNA after 72h transfection. The expression of CTGF and α-SMA significantly decreased in HLECs after transfected by CTGF siRNA for 24h. This effect was not found in negative control siRNA. CONCLUSIONS: SiRNA targeting CTGF decreased CTGF and α-SMA expression in HLECs, which is a potential therapeutic strategy for posterior capsular opacification.

Purpose: Introducing the effect of RNAi in fungi to downregulate essential genes has made it a powerful tool to investigate gene function, with potential strategies for novel disease treatments. Thus, this study is an endeavor to delve into the silencing potentials of siRNA on cyp51A and MDR1 in voriconazole-resistant Aspergillus flavus as the target genes. Methods: In this study, we designed three cyp51A-specific siRNAs and three MDR1-specific siRNAs and after the co-transfection of siRNA into Aspergillus flavus, using lipofectamine, we investigated the effect of different siRNA concentrations (5, 15, 25, 50nM) on cyp51A and MDR1 expressions by qRT-PCR. Finally, the Minimum Inhibitory Concentrations (MICs) of voriconazole for isolates were determined by broth dilution method. Results: Cyp51A siRNA induced 9, 22, 33, 40-fold reductions in cyp51A mRNA expres­sion in a voriconazole-resistant strain following the treatment of the cells with concentrations of 5, 15, 25, 50nM siRNA, respectively. Identically, the same procedure was applied to MDR1, even though it induced 2, 3, 4, 10-fold reductions. The results demonstrated a MIC for voriconazole in the untreated group (4µg per ml), when compared to the group treated with cyp51A-specific siRNA and MDR1-specific siRNA, both at concentrations of 25 and 50nM, yielding 2µg per ml and 1µg per ml when 25 nM was applied and 2µg per ml and 0.5µg per ml when the concentration doubled to 50 nM. Conclusion: In this study, we suggested that siRNA-mediated specific inhibition of cyp51A and MDR1 genes play roles in voriconazole-resistant A.flavus strain and these could be apt target genes for inactivation. The current study promises a bright prospect for the treatment of invasive aspergillosis through the effective deployment of RNAi and gene therapy.

We describe a method by which early developing vasculature can be gene-manipulated independently of the heart in a spatio-temporally controlled manner. Lipofectamine 2000 or 3000, an easy-to-use lipid reagent, has been found to yield a high efficiency of transfection when co-injected with GFP DNA within a critical range of lipid concentration. By exploiting developmentally changing patterns of vasculature and blood flow, we have succeed in controlling the site of transfection: injection with a lipid-DNA cocktail into the heart before or after the blood circulation starts results in a limited and widely spread patterns of transfection, respectively. Furthermore, a cocktail injection into the right dorsal aorta leads to transgenesis of the right half of embryonic vasculature. In addition, this method combined with the siRNA technique has allowed, for the first time, to knockdown the endogenous expression of VE-cadherin (also called Cdh5), which has been implicated in assembly of nasant blood vessels: when Cah5 siRNA is injected into the right dorsal aorta, pronounced defects in the right half of vasculature are observed without heart defects. Whereas infusion-mediated gene transfection method has previously been reported using lipid reagents that were elaborately prepared on their own, Lipofectamine is an easy-use reagent with no requirement of special expertise. The methods reported here would overcome shortcomings of conventional vascular-transgenic animals, such as mice and zebrafish, in which pan-endothelial enhancer-driven transgenesis often leads to the heart malformation, which, in turn, indirectly affects peripheral vasculature due to flow defects. Since a variety of subtypes in vasculature have increasingly been appreciated, the spatio-temporally controllable gene manipulation described in this study offers a powerful tool to understand how the vasculature is established at the molecular level. • Blood flow-mediated transfection enables site-specific transgenesis in vessels. • This transfection technique allows local knockdown of endogenous gene(s) by siRNA. • Knockdown of endogenous VE-cadherin causes vascular defects without heart failure. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the effect of p21Cip1 on survivin transcription levels in breast carcinoma, and to investigate the potential mechanisms. Methods: Epirubicin, a p21Cip1 activator, was used to treat MCF7 cells. Under the action of normal biological functions of p53, pEGFP-C2-p21 was transfected into MCF7 cells by lipofectamine and positive clones were screened out with G418. The expression levels of p21cip1, p53 and survivin mRNA were quantitated by real-time fluorescent polymerase chain reaction (RQ-PCR). MTT assay was utilized to measure cellular viability and proliferation after transfection. Flow cytometry was employed to determine the cell cycle. Hoechst 33342 staining was carried out to assess cell apoptosis. Lastly, several transcription factor sites located at the promoter region of survivin gene, such as, sp1 site, E2F site and p300/CBP, were measured by p21 overexpression using RT-PCR. Results: Following epirubicin treatment, within 24 h, the expression levels of endogenous p21cip1 and p53 were up-regulated, whereas that of survivin was down-regulated. After transfection treatment, p21 inhibited the proliferation of MCF7 cells on days 3 and 4, and MCF7 cells overexpressed p21 mRNA, whereas the level of survivin mRNA in MCF7-p21 groups was markedly down-regulated relative to control group, but overexpression of p21 was not sufficient to cause changes in p53 gene expression. The overexpressed p21 resulted in G1/G0 phase arrest based on cell cycle analysis, but apoptosis was not induced. In addition, co-transcription factors E2F-1, sp1 and p300/CBP mRNA levels decreased significantly compared with normal p21 expression groups. Conclusion: P21cip1 may down-regulate the expression of survivin gene partially by inhibiting the expression level of HAT. [ABSTRACT FROM AUTHOR]

The commercial transfection reagent Lipofectamine has been widely used for cytoplasmic delivery of nucleic acids and for cytosolic engagement with intracellular innate immune sensors to trigger type I interferon (IFN) production. However, the effect of Lipofectamine alone on type I IFN response has not been studied in detail. Here, we show that Lipofectamine induced type I IFN signaling in both RAW 264.7 macrophage‐like cells and primary bone marrow‐derived macrophages. Type I IFN induction was dependent on interferon regulatory factor (IRF)3 and IRF7 and partially required the toll/interleukin‐1 receptor‐domain‐containing adapter‐inducing interferon‐β. In contrast, the transfection reagent Xfect did not activate type I IFN signaling. Our study highlights the potential confounding experimental interpretation when using Lipofectamine‐based transfection for delivering intracellular ligands and provides important insights into lipid signaling in innate immune responses. We found that the transfection reagent Lipofectamine can induce type I interferon signaling in macrophages. [ABSTRACT FROM AUTHOR]

Objective: Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few reports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for transient expression of green fluorescent protein in two genetically different hESC lines, Royan H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after transfection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene. Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 μg and the density of the subjected cells (5×105 and 1×106 cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion: Electroporation was an efficient tool for genetic engineering of hESCs compared to the chemical method. The genetic background of the subjected cell line for transfection seemed to be a fundamental factor in each gene delivery method. For each cell line, optimum voltage rate should be calculated as it has been shown to play a crucial role in cell death and rate of gene delivery.

T-cells play critical roles in various immune reactions, and genetically engineered T-cells have attracted attention for the treatment of cancer and autoimmune diseases. Previously, it is shown that a polyamidoamine dendrimer of generation 4 (G4), modified with 1,2-cyclohexanedicarboxylic anhydride (CHex) and phenylalanine (Phe) (G4-CHex-Phe), is useful for delivery into T-cells and their subsets. In this study, an efficient non-viral gene delivery system is constructed using this dendrimer. Ternary complexes are prepared using different ratios of plasmid DNA, Lipofectamine, and G4-CHex-Phe. A carboxy-terminal dendrimer lacking Phe (G3.5) is used for comparison. These complexes are characterized using agarose gel electrophoresis, dynamic light scattering, and ζpotential measurements. In Jurkat cells, the ternary complex with G4-CHex-Phe at a P/COOH ratio of 1/5 shows higher transfection activity than other complexes, such as binary and ternary complexes with G3.5, without any significant cytotoxicity. The transfection efficiency of the G4-CHex-Phe ternary complexes decreases considerably in the presence of free G4-CHex-Phe and upon altering the complex preparation method. These results suggest that G4-CHex-Phe promotes the cellular internalization of the complexes, which is useful for gene delivery into T-cells., (© 2023 Wiley-VCH GmbH.)

Background and Objectives: Nonviral carriers including those based on synthetic cationic lipids, offer several advantages over the viral counterparts. These carriers are able to form complexes with nucleic acids and deliver genes into the cells via the cellular endocytosis pathway, without significant toxicity. The level of transgenes expression depends on some experimental variables including cell type and density, Lipofectamine and DNA concentrations and Lipofectamine-DNA complexing time. The main objective of this study was to optimize transfection of SW480 colon cancer cells with Lipofectamine 2000. Methods: In this study, SW480 cells were transfected with plasmid containing green fluorescent protein reporter gene using Lipofectamine 2000. Green fluorescent protein expression was studied under a reverse fluorescence microscope and the results were analyzed with the ImageJ software. Effect of different quantities of plasmid DNA and different Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Results: The optimal volume of Lipofectamine and quantity of plasmid was 2 μl and 1μg, respectively, which showed 59% efficiency for the transfection of SW480 cells at 24 hours post-transfection. Conclusion: This study shows that Lipofectamine 2000 is an efficient reagent for the delivery of genes into SW480 cells. According to the results, the quantity of DNA per transfection and reagent concentrations are essential factors for a successful transfection. [ABSTRACT FROM AUTHOR]

Background Allelic heterogeneity is an important feature of the GLA gene for which almost 900 known genetic variants have been discovered so far. Pathogenetic GLA variants cause alpha-galactosidase A (α-Gal A) enzyme deficiency leading to the X-linked lysosomal storage disorder Fabry disease (FD). Benign GLA intronic and exonic variants ( e.g. pseudodeficient p.Asp313Tyr) have also been described. Some GLA missense variants, previously deemed to be pathogenetic ( e.g. p.Glu66Gln and p.Arg118Cys), they have been reclassified as benign after re-evaluation by functional and population studies. Hence, the functional role of novel GLA variants should be investigated to assess their clinical relevance. Results We identified six GLA variants in 4 males and 2 females who exhibited symptoms of FD: c.159C>G p.(Asn53Lys), c.400T>C p.(Tyr134His), c.680G>C (p.Arg227Pro), c.815A>T p.(Asn272Ile), c.907A>T p.(Ile303Phe) and c.1163_1165delTCC (p.Leu388del). We evaluated their impact on the α-Gal A protein by bioinformatic analysis and homology modelling, by analysis of the GLA mRNA, and by site-directed mutagenesis and in vitro expression studies. We also measured their responsiveness to the pharmacological chaperone DGJ. Conclusions The six detected GLA variants cause deficient α-Gal A activity and impairment or loss of the protein wild-type structure. We found p.Asn53Lys and p.Ile303Phe variants to be susceptible to DGJ. [ABSTRACT FROM AUTHOR]

Objective: In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry.Results: The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p < 0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~ 83.94 and ~ 77.01% for HEK-293T), and cancer cells (~ 71.69 and ~ 67.19% for TC-1, and ~ 73.86 and ~ 67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~ 60-80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~ 63.66%) compared to TurboFect (~ 32.95%); however, the efficiency level of hPP10 was only ~ 17.51 and ~ 16.36% in TC-1 and A549 cells.Conclusions: Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers. [ABSTRACT FROM AUTHOR]

Objective: An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent.Study Design: In vitro.Methods: We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-β cascade implicated in fibroplasia in the vocal folds and other tissues.Results: The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P < 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (P < 0.05). Suppression was more dose-sensitive with lipitoid. At a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a five-fold increase in lipitoid concentration. Increased number of administrations enhanced gene suppression, ∼45% decrease between one and four administrations. Neither serum nor media type altered efficiency.Conclusion: Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility.Level Of Evidence: NA. Laryngoscope, 127:E231-E237, 2017. [ABSTRACT FROM AUTHOR]

Purpose: Introducing the effect of RNAi in fungi to downregulate essential genes has made it a powerful tool to investigate gene function, with potential strategies for novel disease treatments. Thus, this study is an endeavor to delve into the silencing potentials of siRNA on cyp51A and MDR1 in voriconazole-resistant Aspergillus flavus as the target genes. Methods: In this study, we designed three cyp51A-specific siRNAs and three MDR1-specific siRNAs and after the co-transfection of siRNA into Aspergillus flavus, using lipofectamine, we investigated the effect of different siRNA concentrations (5, 15, 25, 50nM) on cyp51A and MDR1 expressions by qRT-PCR. Finally, the Minimum Inhibitory Concentrations (MICs) of voriconazole for isolates were determined by broth dilution method. Results: Cyp51A siRNA induced 9, 22, 33, 40-fold reductions in cyp51A mRNA expression in a voriconazole-resistant strain following the treatment of the cells with concentrations of 5, 15, 25, 50nM siRNA, respectively. Identically, the same procedure was applied to MDR1, even though it induced 2, 3, 4, 10-fold reductions. The results demonstrated a MIC for voriconazole in the untreated group (4μg per ml), when compared to the group treated with cyp51A-specific siRNA and MDR1-specific siRNA, both at concentrations of 25 and 50nM, yielding 2μg per ml and 1μg per ml when 25 nM was applied and 2μg per ml and 0.5μg per ml when the concentration doubled to 50 nM. Conclusion: In this study, we suggested that siRNA-mediated specific inhibition of cyp51A and MDR1 genes play roles in voriconazole-resistant A.flavus strain and these could be apt target genes for inactivation. The current study promises a bright prospect for the treatment of invasive aspergillosis through the effective deployment of RNAi and gene therapy. [ABSTRACT FROM AUTHOR]