Green Fluorescent Protein Transfection Research Articles

Chitosan-Functionalized Poly(β-Amino Ester) Hybrid System for Gene Delivery in Vaginal Mucosal Epithelial Cells.

Gene therapy displays great promise in the treatment of cervical cancer. The occurrence of cervical cancer is highly related to persistent human papilloma virus (HPV) infection. The HPV oncogene can be cleaved via gene editing technology to eliminate carcinogenic elements. However, the successful application of the gene therapy method depends on effective gene delivery into the vagina. To improve mucosal penetration and adhesion ability, quaternized chitosan was introduced into the poly(β-amino ester) (PBAE) gene-delivery system in the form of quaternized chitosan-g-PBAE (QCP). At a mass ratio of PBAE:QCP of 2:1, the polymers exhibited the highest green fluorescent protein (GFP) transfection efficiency in HEK293T and ME180 cells, which was 1.1 and 5.4 times higher than that of PEI 25 kD. At this mass ratio, PBAE-QCP effectively compressed the GFP into spherical polyplex nanoparticles (PQ-GFP NPs) with a diameter of 255.5 nm. In vivo results indicated that owing to the mucopenetration and adhesion capability of quaternized CS, the GFP transfection efficiency of the PBAE-QCP hybrid system was considerably higher than those of PBAE and PEI 25 kD in the vaginal epithelial cells of Sprague-Dawley rats. Furthermore, the new system demonstrated low toxicity and good safety, laying an effective foundation for its further application in gene therapy.

Optimization of transfection methods for human lymphoblast TK6 cell line

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

Controlled Differentiation of Mesenchymal Stem Cells into Hyaline Cartilage in miR-140-Activated Collagen Hydrogel.

Hypertrophic cartilage formation is a major setback in mesenchymal stem cells (MSCs)-mediated cartilage repair, and overcoming it requires optimization of differentiation. Here, we tested the miR-140 activated collagen hydrogel for the chondrogenic differentiation of MSCs and to produce hyaline cartilage. Bone marrow MSCs isolated from 3 patients were pretreated with miR-140 and then chondrogenic differentiated. The 3-dimensional (3D) transfection potential of 5 different transfection reagents (Polyethylenimine, Lipofectamine, TransIT-X2, Amide:Cholesterol-based liposomes [AmC] and AmC pegylated with Tocofersolan [AmCTOC]) was compared and the reagent that showed higher green fluorescent protein (GFP) expression was selected. Finally, the collagen hydrogel was activated using miR-140-transfection complex and sustained delivered to MSCs during chondrogenic differentiation. After differentiation, the outcome was assessed by reverse transcription-polymerase chain reaction (RT-PCR), histology, immunohistochemistry, and compared with scrambled miRNA treated control. Pretreatment of MSCs with miR-140 significantly increased the expression of cartilage-specific genes (COL2A1, SOX9, and ACAN) with reduced hypertrophic chondrocyte (COL10A1) marker expression and better safranin-O staining than the control. The AmCTOC liposome showed a significant increase in 3D transfection of GFP expressing plasmid than the others. Furthermore, the knockdown of GAPDH using siRNA in HEK cells and expression of GFP mRNA in human bone marrow MSCs confirmed the 3D-transfection efficiency of AmCTOC. The sustained delivery of miR-140 using activated matrix formed a hyaline cartilage-like tissue with minimal COL10A1 expression in RT-PCR and immunohistochemistry. Our results demonstrated the therapeutic potential of miR-140-activated hydrogel for MSCs-based cartilage tissue engineering, which could also be used for endogenous stem cells-mediated cartilage repair.

Expression of transduced nucleolin promotes the clearance of accumulated α-synuclein in rodent cells and animal model

Alpha-synuclein (αSyn) is a major component of Lewy bodies, which are a known pathogenic marker of Parkinson's disease (PD). The dysfunction of protein degradation machinery causes αSyn accumulation. The reinforcement of αSyn degradation is a potential therapeutic target for PD because accumulated αSyn is responsible for the pathogenesis of PD. Nucleolin (NCL) is essential in the formation of the nucleolar structure. The function of NCL is correlated with oxidative stress-mediated cell death. A previous study demonstrated that NCL overexpression alleviated rotenone-induced neurotoxic effects, whereas knockdown of NCL had the opposite effect. These results suggest that NCL malfunction would exacerbate PD pathology. Thus, it was hypothesized that the introduction of ectopic NCL could rescue α-synucleinopathy in PD. This study investigated whether the ectopic expression of NCL facilitates αSyn clearance. Ectopic expression of NCL was accomplished via the transfection of green fluorescent protein (GFP) or GFP-NCL in mouse embryonic fibroblasts (MEF) or transduction of GFP or GFP-NCL using lentivirus in rat primary cortical neurons and mouse substantia nigra. NCL overexpression enhanced the clearance of accumulated or aggregated αSyn in MEFs and rat primary cortical neurons. The activity of the autophagy-lysosome pathway was enhanced by NCL expression. NCL transduction in the substantia nigra, which was co-injected with αSyn fibrils, rescued PD manifestation. The elevation of NCL levels may reflect a therapeutic strategy for α-synucleinopathy in PD.

Impact of photobiomodulation using four diode laser wavelengths of on cationic liposome gene transfection into pre-osteoblast cells

Gene therapy can be an effective treatment modality for some severe genetic diseases. Despite efforts to improve their performance, non-viral gene delivery methods remain inefficient and costly. As an alternative to viral vectors, cationic liposomes have a good safety profile and low immunogenicity, but relatively low transfection efficiency. They may also be toxic to cells at high concentrations. Given these challenges, the present study explored the impact of photobiomodulation (PBM) on cationic liposome plasmid DNA transfection in terms of its efficiency and toxicity, using Lipofectamine 2000 to carry green fluorescent protein (GFP) encoding plasmid DNA, with the pre-osteoblast MC3T3-E1 cell line as the target. Cultures were irradiated using diode lasers (445, 685, 810, or 970 nm) at 200 mW using pulsed mode (50 Hz), with a power density of 104.64 mW/cm2, and irradiance from 6 to 18 joules. To determine transfection efficiency, expression of GFP was assessed using confocal laser scanning microscopy and flow cytometry. Cell viability was evaluated using the MTT assay. PBM using 810 nm and 970 nm lasers significantly enhanced transfection efficiency for GFP, indicating more efficient uptake of plasmid DNA. Conversely, laser irradiation at 445 nm and 685 nm wavelengths reduced the GFP transfection efficiency. Treatment using 685, 810, and 970 nm lasers at 12 J maintained cell viability and prevented toxicity of cationic liposomes. Overall, these findings support the concept that PBM using near infrared laser wavelengths can enhance transfection efficiency and support cell viability when cationic liposomes are used as the vector in gene therapy.

Lipodendriplexes mediated enhanced gene delivery: a cellular to pre-clinical investigation

Clinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (–NH2) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.

Nanoparticle‐Mediated Suicide Gene Therapy for Triple Negative Breast Cancer Treatment

AbstractSystemic chemotherapy has not significantly reduced clinical demand for triple negative breast cancer (TNBC) treatments. To address the need for more effective therapy, the use of nonviral nanoparticles is explored to deliver suicide gene therapy as valuable alternative to protect nucleic acids in the bloodstream and improve their tumor uptake. Biocompatible cationic lipid nanoparticles are developed as a novel delivery system of a suicide plasmid gene encoding saporin. Active targeting is accomplished by taking advantage of nanoparticle functionalization with U11 peptide, designed to be directed toward urokinase plasminogen activator receptor, limiting off‐target toxicity. The antitumor effect of U11‐lipid‐protamine‐DNA (U11‐LPD) nanoparticles are tested in TBNC cells, showing a strong prevalence of targeted versus nontargeted nanoparticles in terms of uptake kinetics and proliferation inhibition. Transfection of green fluorescent protein (GFP) plasmid in MDA‐MB‐231 cells is demonstrated. U11‐LPD nanoparticles administered by retro bulbar injection exhibit excellent tumor tropism in TNBC orthotopic xenograft mice and effectively transfect TNBC cells with saporin plasmid resulting in tumor mass reduction. No systemic toxicity or organ damage is discovered after repeated treatments with nanoparticles. The findings suggest that systemic administration of targeted LPD nanoparticles to deliver saporin safely allows for active inhibition of cancer progression even in the absence of specific promoter gene sequences.

The Addition of GFP to Exosomes Influences Cardioprotective Potential Through an RNA Binding Mechanism

Exosomes are 40–100nm vesicles that have been extensively investigated for their therapeutic potential. Exosomes isolated from mesenchymal stem cells (MSCs) are shown to have cardioprotective potential both in models of infarction and heart failure. Our previous work supports that this cardioprotection occurs through the delivery of microRNAs (specifically, mir21a‐5p). We observed that with the addition of a green fluorescent protein (GFP), the protective potential of MSC derived exosomes decreases. In order to better understand this observed effect, we addressed the hypothesis that the modification of MSCs with GFP limits the cardioprotective potential of the exosomes by binding cardioprotective RNA and limiting its functional delivery to target cells.Extracellular vesicles (EVs) were isolated via ultracentrifugation from C57BL/6 derived MSCs that are transduced with GFP producing plasmids. Confirmation of exosomal content was done via exosomal protein marker analysis (LAMP1, CD63) with Amnis Imaging Flowcytometry. Populations in the GFP containing MSC‐derived exosomes showed detectable GFP signal and were validated to contain exosomal protein markers. GFP protein was immunoprecipitated from isolated EVs. The Qubit Fluorometer and Agilent 2100 Bioanalyzer were used to assess the integrity and concentration and indicated that the RNA is measurable in MSC derived EVs (both GFP and wild type). The isolated miRNA was prepared into libraries of template molecules and sequenced on the Illumina MiSeq. Analyses of the derived data show that miRNA components of EVs vary with the addition of GFP. However, miR21a‐5p was shown to be the most abundant in both wild type (WT) and GFP containing groups. Functionally, RNA delivery via GFP containing exosomes into H9c2 cardiomyoblasts does not show protection against cell death in a simulated ischemia/reperfusion model. In vivo murine models also indicate that exosomes isolated from GFP‐MSCs do not confer protection against left anterior descending coronary artery (LAD) occlusion‐induced myocardial infarction, as exosomes isolated from WT‐MSCs were shown to do. Further, real‐time RT‐PCR showed that miR‐21a‐5P is present in GFP‐associated RNA.Analysis of MiSeq data from WT and GFP‐MSC exosomes showed no major differences in miR‐21a‐5P levels. However, after purification of GFP‐associated RNA, we found that this RNA contains miR‐21a‐5P, suggesting that at least some amount of this miR is bound to GFP in the GFP‐containing exosomes. Future work will establish the profiles of miRNAs associated with GFP in order to develop a profile of cardioprotective exosomal content, and better understand the potential for GFP binding and transfection of miRNAs.Support or Funding InformationThis work was supported by the National Institutes of Health under award number R01HL091478 to WK Jones.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti-cancer efficacy of biotinylated chitosan nanoparticles in liver cancer.

The present study investigated the synthesis of biotinylated chitosan (Bio-CS) from chitosan using a nanomaterial skeleton with biotin and the successful targeting of the formulation in liver cancer cells. Bio-CS was validated by fourier transformed infrared spectroscopy and hydrogen-1 nuclear magnetic resonance spectroscopy. Bio-CS and plasmid DNA were used to construct Bio-CS/plasmid DNA nanoparticles according to the optimal molar ratio of 1:1 and the optimal pH-value of 5.5. Under these conditions, the parameters mean particle size, potential, encapsulation rate and drug loading, were 82.9 nm, +21.8 mV, 85.7% and 35.4%, respectively. Bio-CS exhibited an apparent liver cancer targeting effect in vitro and in vivo, as demonstrated by confocal laser scanning, green fluorescent protein transfection, and in vivo imaging assays. In addition, the Bio-CS/plasmid DNA nanoparticles significantly increased the survival period of the orthotropic liver cancer mouse model compared with the plasmid DNA, with no apparent side effects on the cells. Bio-CS nanomaterials stimulated an immune response in hepatoma cells via increased expression of GM-CSF, IL-21 and Rae-1 markers. The data suggest that Bio-CS increased the inhibition of liver cancer cell proliferation in vitro and the activation of the cellular immunity in vivo.

An optimized gene transfection system in WERI-Rb1 cells.

The pathogenesis of Rb1 gene inactivation indicates that gene therapy could be a promising treatment for retinoblastoma. An appropriate gene transfer system is the basis for successful gene therapy; however, little attention has been given to an effective gene transfer system for retinoblastoma therapy in previous studies. This study was designed to provide an optimized transgene system for WERI-Rb1 cells (W-RBCs). Green fluorescent protein (GFP) was adopted as a reporter. Four classic viral vectors based on retroviruses, recombinant adeno-associated viruses (rAAV2, rAAV2/1), lentiviruses (LVs) and a novel non-viral vector X-treme HP reagent were adopted for W-RBC gene transfection. The efficacy and cytotoxicity were comprehensively compared among the different vectors through GFP expression and the trypan blue exclusion test. Furthermore, the serum and cell culture status were also optimized for better transfection. Cells transfected by rAAV2/1 expressed more GFP protein and exhibited less staining with trypan blue, compared to the rAAV2 counterpart. However, in comparison to the retroviral group, both the rAAV2/1 and LV groups had considerably less GFP+ cells. Interestingly, the X-treme HP presented a similar GFP transfection capacity to the retroviral vector, but with a much lower cytotoxicity. Furthermore, there were more GFP+ cells in a suspended condition than that in an adherent culture. Moreover, cells in a serum-positive system expressed more GFP, while cells in a serum-free system showed lower GFP expression and higher cytotoxicity. In conclusion, the retroviral vector and the X-treme HP are effective for W-RBC gene transfection, while the X-treme HP is more preferable due to its lower cytotoxicity. Moreover, the suspended cell culture system is superior to the adherent system, and the serum protects cell viability and facilitates the gene transfection of W-RBCs. This study presents an effective, convenient, and low toxic transfection system for gene delivery in W-RBCs and provides a promising system for further gene therapy of retinoblastoma.

Effect of indianhedgehog gene transfection into rabbit bone marrow mesenchymal stem cells in promoting chondrogenic differentiation and inhibiting cartilage aging in rotary cell culture system

?To investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. ?The 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen type Ⅱ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ?After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P<0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P<0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P<0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen type Ⅱ and ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P<0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen type Ⅱ protein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P<0.05); RCCS 1 group expressed high levels of collagen type Ⅱ and ANCN proteins at all stages (P<0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ?Under the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

Sacasg and sacalg: new GFP-labelled camel skin and lung fibroblast cell lines

Camelids skin is claimed to respond differently under different physiological and pathological condition, however, there is no in vitro model with a reporter to investigate this claim. So generating camel fibroblast cell lines that expresses green fluorescent protein (GFP) would be important as a tool to monitor camel cell growth, migration and other processes. Hence, in this investigation, we created a GFP expressing fibroblast cell lines derived from a primary skin and lung fibroblast cell lines of the Arabian camel with a stable expression of GFP which was transfected using the pCAG-EGFP plasmid with CMV enhancer via Lipofectamine 2000 reagent. Stably transfected clones were selected by puromycin screening. The results revealed that camel fibroblasts can be efficiently transduced in vitro using pCAG-CMV-based vectors and that these vectors direct long-term transgene expression without evident toxicity, pathogenesis or alteration of native fibroblast morphology. Immunofluorescence staining showed no differences in the expression of fibroblast biomarkers between the transfected and non-transfected cell lines. The viability of thawed cells remained above 85% after cryopreservation in liquid nitrogen. The gene expression of the fibroblast markers was not different in the transfected cell lines. Taken together, we have established and fully characterised GFP expressing-fibroblast cell lines of Arabian camel. Based on our assays, we conclude that transfection of GFP into the Arabian camel skin and lung fibroblasts did not change their observed properties. The GFP-labelled cell lines may represent a new tool for convenient monitoring of live primary camel fibroblasts.

2D45 細胞骨格の変形分布とカルシウム応答の同時計測による細胞の力覚システムの検討

This paper describes a novel method to investigate a cell mechanosensing system and the quantitative relationship between the deformation of cytoskeletal structure and the change of intracellular calcium ion concentration as biochemical response in a living cell stimulated by a micropipette. Gene transfection of green fluorescent protein enabled visualization of actin cytoskeleton in cells. Local deformation was applied to a cell by a micropipette and the displacement distribution of actin cytoskeleton in the whole cell was automatically obtained from the two images of the cell before and after deformation by using Kanade-Lucas-Tomasi method which is one of the feature tracking algorithms. Intracellular calcium ion response to the same mechanical stimulation was measured as the spatial and temporal changes of intensity of a fluorescent marker loaded to osteoblasts. These measurement were simultaneously conducted in order to clarify the mechanism of converting mechanical deformation into calcium signaling in a cell. As a result, the change of intracellular calcium ion concentration increases with increasing the displacement of actin cytoskeleton and calcium ion response starts in the area of large cytoskeletal displacement. These indicate that the deformation distribution of actin cytoskeleton is highly related to the cell mechanosensing system.

Small hydrophobe substitution on polyethyleneimine for effective plasmid DNA delivery

Event Abstract Back to Event Small hydrophobe substitution on polyethyleneimine for effective plasmid DNA delivery Bindu Thapa1, Samarwadee Plianwong2, Remant Kc3, Bradely Rutherford3 and Hasan Uludag1, 3, 4 1 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada 2 Silpakorn University, Faculty of Pharmacy, Thailand 3 University of Alberta, Department of Chemical and Material Engineering, Canada 4 University of Alberta, Department of Biomedical Engineering, Canada Introduction: Improved delivery systems are needed for intracellular delivery of sensitive biologics such as plasmid DNA. Cationic polymers have been developed for this end due to their safety and facile chemistry to tailor their properties. These polymers complex with DNA via electrostatic interactions, and condense them into compact nano-sized polyplexes. Chemical modification of low molecular weight PEIs with hydrophobic groups improves delivery due enhanced cellular uptake and unpacking efficacy of the polyplexes. Unlike bulky lipids, the impact of small hydrophobes on cationic polymers for DNA delivery has not been explored in detail; it is possible that small hydrophobes might be superior substituents due to better grafting reactions and unique properties. In this study, we modified 1.2 kDa PEIs using small hydrophobe propionate (PrA; CH2-CH3) and extensively explored the features of resultant DNA polyplexes and delivery efficiency. Materials and Methods: The PrA grafted 1.2 PEI (PEI-PrA; Figure 1) was synthesized via N-acylation and characterized through 1H-NMR spectroscopy[1]. DNA binding efficacy of PEI1.2-PrAs were investigated through agarose gel electrophoresis and size-surface charge through dynamic light scattering[2]. Cellular toxicity, DNA uptake (with Cy3-labeleld DNA) and transfection efficacy of PEI1.2-PrA/DNA complexes was studied in breast cancer cells MDA-231 and MCF-7 as a function of PrA substitution amount, post transfection time and polymer:DNA ratio for complex formation. Green fluorescent protein (GFP) transfection was assessed by flow cytometry. Results and Discussions: Substitution efficacy of PrA onto 1.2 PEI was increased with feed ratio (PrA/PEI) with highest grafting of 1.6 PrA/PEI. DNA binding efficacy of PEI-PrA was decreased in proportion with PrA substitution as a consequences of –NH2 consumption and/or steric hindrances. Hydrodynamic size of polyplexes was identical irrespective to PrA substitution, but surface charge initially increased with PrA substitution and later decreased at high PrA substitutions. Cellular toxicity of the polymers was increased with PrA substitution but the polymers still displayed less toxicity compared to 25 kDa PEI (PEI25). GFP transfection efficiency in both MDA-231 and MCF-7 was significantly increased with optimal PrA substitution (0.5-1 PrA/PEI) while polymers with the highest substitution and parent polymer were ineffective (Figure 2). These results paralleled the results obtained from cellular uptake of plasmid DNA. Importantly, transfection efficiency of PEI-PrA1 (PrA/PEI = 0.76) was higher than long chain lipid (C=18) grafted 1.2 PEI and comparable to PEI25. In addition, PEI-PrA1 showed higher transfection than PEI 25 at day 7 and 14, showing a relatively stable transfection in MDA-231 cells. Conclusions: Substitution of small hydrophobe PrA onto PEI1.2 enhanced transfection efficiency in breast cancer cells, but excess substitution (>1.2 PrA/PEI) was detrimental, emphasizing the importance of balancing polymer hydrophobicity for effective gene delivery. Thus, integration of small hydrophobe onto low molecular weight PEIs seems as effective as large lipids. This study was supported by an NCPRM (NSERC CREATE program for regenerative medicine)

Size-dependent gene delivery of amine-modified silica nanoparticles

Silica-based nanoparticles are promising carriers for gene delivery applications. To gain insights into the effect of particle size on gene transfection efficiency, amine-modified monodisperse Stober spheres (NH2-SS) with diameters of 125, 230, 330, 440, and 570 nm were synthesized. The in vitro transfection efficiencies of NH2-SS for delivering plasmid DNA encoding green fluorescent protein (GFP) (pcDNA3-EGFP, abbreviated as pcDNA, 6.1 kbp) were studied in HEK293T cells. NH2-SS with a diameter of 330 nm (NH2-SS330) showed the highest GFP transfection level compared to NH2-SS particles with other sizes. The transfection efficiency was found as a compromise between the binding capacity and cellular uptake performance of NH2-SS330 and pcDNA conjugates. NH2-SS330 also demonstrated the highest transfection efficiency for plasmid DNA (pDNA) with a bigger size of 8.9 kbp. To our knowledge, this study is the first to demonstrate the significance of particle size for gene transfection efficiency in silica-based gene delivery systems. Our findings are crucial to the rational design of synthetic vectors for gene therapy.

20722 構造変形と生化学応答の同時計測による細胞の力覚システムの検討

This paper describes the relationship between the deformation of cytoskeletal structure and the change in calcium ion concentration as biochemical response in a living cell deformed by a micropipette. Gene transfection of green fluorescent protein enabled visualization of actin cytoskeleton in cells. Local deformation was applied to a cell by a micropipette and the displacement distribution of actin in the whole cell was automatically obtained from the two images of the cell before and after deformation by using KLT method which is one of the feature tracking algorithms. Calcium ion response to the same mechanical stimulation was measured as the spatial and temporal changes of intensity of a fluorescent marker loaded to osteoblasts. As a result, displacement of actin and change in calcium ion concentration were highly correlated and calcium ion response time was delayed with increasing the distance from a stimulation point in a cell.

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction.

To investigate adenoviral transduction in mesenchymal stem cells (MSCs) and effects on stemness in vitro and function as a cell therapy in vivo. Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine (PEI)-mediated transfection of pcDNA3-eGFP or adenoviral transduction of green fluorescent protein (GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness (i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2 (BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology. PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs (81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs (78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs (7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression (0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs (1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitro were not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult (P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells (P < 0.05). Ad-BMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs (83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone (1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05). Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.

In vivo tracking of adipose-derived stem cells labeled with green fluorescent protein and luciferase dual reporter gene

Objective T o explore the feasibility of in vivo tracking of adipose-derived stem cells( ADSC s) by using lentiviral vectors containing green fluorescent protein( GFP) and luciferase( Luc) dual reporter gene transfection and bioluminescent imaging techniques. Methods T he ADSC s were isolated from human adipose tissue samples of cosmetic subdermal liposuction. T he ADSC s were transfected by lentiviral vectors containing GFP and Luc. T he labeling efficiency was evaluated by fluorescence microscope and flow cytometry in vitro. T he transfected ADSC s were subcutaneously injected into BALB /C mice, and then a non-invasive bioluminescence imaging procedure was performed 1 h, 1, 2, 4, 6 and 8 weeks after transplantation. T he DAPI staining of skin tissues was performed to identify the survival and the location of transplanted ADSC s. Results High GFP expression was observed in transfected ADSC s by lentiviral vector under fluorescence microscope. T he transfection efficiency of lentiviral vectors containing GFP and Luc was 87. 3%. T ransfected ADSC s can express GFP and Luc after 25 successive generations, and the transduction of ADSC s with lentiviral bioluminescence reporter vectors had no effect on cell proliferation. T he bioluminescence of ADSC s in vivo can be detectable as long as 8 weeks after transplantion, and the ADSC s mainly survive in the inoculating site of the thigh.Histological analysis of sections showed that the labeled ADSC s located in the subcutaneous layer of the implantation site. Conclusion U sing lentiviral vectors containing GFP and Luc dual reporter gene transfection and bioluminescent imaging techniques can monitor ADSC s in vivo.

EZH2 promotes tumor progression by increasing VEGF expression in clear cell renal cell carcinoma.

The present study is to evaluate the expression level of enhancer of zeste homolog 2 (EZH2) and vascular endothelial growth factor (VEGF), and analyze their correlations with clinicopathological characteristics and survival in patients with clear cell renal cell carcinoma (CCRCC). The effect of EZH2 on apoptosis and cell proliferation in 786-O renal cancer cell line is investigated. The expression level of EZH2 and VEGF was detected in 185 primary CCRCC patients' tissues using tissue microarray and immunohistochemistry. Small interfering RNA or enhanced green fluorescent protein transfection was employed to investigate the effect of EZH2 inhibition or overexpression on VEGF expression, apoptosis and cell proliferation in 786-O cells using flow cytometry, immunofluorescence microscopy, quantitative real-time reverse-transcription polymerase chain reaction and Western blot analysis. High expression level of EZH2 and VEGF was observed in advanced CCRCC and correlated with the TNM stage (p=0.013, p=0.001) and distant metastasis (p=0.011, p=0.038), respectively. EZH2 was positively correlated with VEGF in CCRCC tissues (correlation coefficient=0.850, p<0.001). Kaplan-Meier survival analysis revealed that patients with positive EZH2 expression had a shorter overall survival time compared to patients with negative EZH2 expression (34.3 vs. 67.2, p<0.001). In 786-O cells, EZH2 silencing inhibited VEGF expression and cell proliferation while increasing apoptosis (p<0.001). EZH2 overexpression promoted VEGF expression and cell proliferation while inhibiting apoptosis (p<0.001). EZH2 correlates positively with VEGF and associates with adverse clinicopathologic characteristics and shorter survival time in CCRCC patients. EZH2 accelerates antiapoptosis and cell cycle in 786-O cells.

Lignin Nanotubes As Vehicles for Gene Delivery into Human Cells

Lignin nanotubes (LNTs) synthesized from the aromatic plant cell wall polymer lignin in a sacrificial alumina membrane template have as useful features their flexibility, ease of functionalization due to the availability of many functional groups, label-free detection by autofluorescence, and customizable optical properties. In this report we show that the physicochemical properties of LNTs can be varied over a wide range to match requirements for specific applications by using lignin with different subunit composition, a function of plant species and genotype, and by choosing the lignin isolation method (thioglycolic acid, phosphoric acid, sulfuric acid (Klason), sodium hydroxide lignin), which influences the size and reactivity of the lignin fragments. Cytotoxicity studies with human HeLa cells showed that concentrations of up to 90 mg/mL are tolerated, which is a 10-fold higher concentration than observed for single- or multiwalled carbon nanotubes (CNTs). Confocal microscopy imaging revealed that all LNT formulations enter HeLa cells without auxiliary agents and that LNTs made from NaOH-lignin penetrate the cell nucleus. We further show that DNA can adsorb to LNTs. Consequently, exposure of HeLa cells to LNTs coated with DNA encoding the green fluorescent protein (GFP) leads to transfection and expression of GFP. The highest transfection efficiency was obtained with LNTs made from NaOH-lignin due to a combination of high DNA binding capacity and DNA delivery directly into the nucleus. These combined features of LNTs make LNTs attractive as smart delivery vehicles of DNA without the cytotoxicity associated with CNTs or the immunogenicity of viral vectors.