Papers by Keyword: Transfection

Abstract: The magnetic Fe₂O₃ nanoparticles were prepared by co-precipitation method with FeCl₃ and NaOH as starting reagents. The surface of Fe₂O₃ nanoparticles was modified with tetraethyl orthosilicate. Fe₂O₃@SiO₂ nanocomposites were calcined at 600 °C. The nanocomposites were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX). The PLL-Fe₂O₃@SiO₂ (SMNP) was prepared by modifying with poly-L-lysine on the surface. The SMNP combined with plasmid siRNA by static electrical charges as one of gene carriers was transfected into SD rat neurons. The results of fluorescence microscope and Prussian blue staining show that SMNP can effectively enter cells. Therefore, SMNP are one kind of novel and effective gene carriers, it can transfect the plasmid which carries the siRNA into SD rats neurons in vitro.

Abstract: In order to explore the related mechanism of Foxp3 in tumor immune escape, the study detected the expression of Foxp3 in lewis lung cancer (LLC) cells and analyzed the expression of TGF-β1 and IL-10 in Foxp3 overexpressed LLC cells. Foxp3 mRNA was detected in LLC cells by RT-PCR. Foxp3 was highly expressed in Foxp3 transfected LLC group than that of in empty vector group and LLC group by RT-PCR(P <0.01). The mRNA and protein expression of TGF-β1 and IL-10 significantly increased in Foxp3 transfected LLC group than that of in empty vector group and LLC group by RT-PCR and ELISA(P <0.05). These results suggest that Foxp3 in LLC cells may promote tumor immune escape by enhancing the expression of TGF-β1 and IL-10.

Abstract: Gene therapy is a widespread and promising treatment of many diseases resulting from genetic disorders, infections and cancer. The feasibility of the gene therapy is mainly depends on the development of appropriate method and suitable vectors. For an efficient gene delivery, it is very important to use a carrier that is easy to produce, stable, non-oncogenic and non-immunogenic. Currently most of the vectors actually suffer from many problems. Therefore, the ideal gene therapy delivery system should be developed that can be easily used for highly efficient delivery and able to maintain long-term gene expression, and can be applicable to basic research as well as clinical settings. This article provides a brief over view on the concept and aim of gene delivery, the different gene delivery systems and use of different materials as a carrier in the area of gene therapy.

Abstract: Polyphosphate is a biocompatible, modified and functionalized biodegradable polymer. It has a broad application prospects in the field of medical biomaterials. It is an excellent drug carrier material. In this essay, through the open-loop and ATRP polymerization, it synthesizes biodegradable amphiphilic triblock copolymer PEG-PCL-PDMAEMA. Composition and structure of the polymer is Characterized by ¹H NMR and IR In this essay. This essay also studies that the polymer in water can be self-assembled to form a stable nanoparticles; The polymer micelle can form composite particles with siRNA,. This essay also studies the load and transfection of the polymer micelles on siRNA.

Abstract: The goal in this paper is to investigate the efficiency of poly-PLL (Poly-L-lysine)/ Alg (Alginate) vector mediated virus genomic DNA transfection and the virus genomic DNA’s biological activity in vivo. After Pseudorabies virus (PRV) genomic DNA being adhered to the porous СаСО3 particles, PLL and Alg were alternately polymerized on the surface of the porous DNA-СаСО₃ particles to 7 layers, which were later dissolved them by EDTA to remove СаСО₃ cores; the vectors in which the DNA were coated by poly-PLL/Alg, were harvested to infect the rabbits and observe the replication of viral DNA. Porous СаСО3 particles, which were obtained from the reaction between Na2CO₃ and CaCl₂, had an efficiency of absorbing DNA 1 µg/mg СаСО₃ particles. After being coated by PLL/Alg, microcapsules were obtained with the diameter of 2-4 µm. 10.0 µg of poly-PLL/Alg-PRV DNA microcapsules could cause rabbits’ death by intramuscular injection. The identification of PCR shows that the death was caused by PRV infection. The results indicate that Poly-PLL/Alg microcapsules can mediate efficient transfection of DNA.

Abstract: Cationic carriers including polyethylenimine, liposomes, and chitosan have been used to transfer plasmid DNA in vitro by condensing anionic DNA. Here, oligochitosan (OC) was found to have capacity for in-vitro gene delivery in four cell lines. Plasmid containing green fluorescent protein (GFP) gene was used as a reporter gene. The transfection efficacy and cell viability of the transfection vehicles were analyzed by using a high-throughput image analyzer. We found that DNA polyplexes formed by high dosage of OC could be efficiently delivered into the cells. The combination of OC and polyethylenimine (PEI) were found to significantly enhance the fluorescence protein expression. The introduction of oligochitosan in PEI-mediated transfection could increase the transfection efficacy and could reduce the toxicity of PEI. Additionally, the synergistic effects of PEI and OC were confirmed in CHO, Caco2, Hep-SK, and 3T3 cell lines. The detailed mechanism of PEI and oligochitosan on transfection was investigated by using gel retardation and DNase degradation experiments. A facile and inexpensive construction of gene delivering vehicles was developed herein by using oligochitosan and PEI.

Abstract: A surface-mediated gene transfer system using DNA-calcium phosphate (CaP) composite layers (D-CaP layers) would be useful in tissue engineeing. In previous studies, D-CaP layers were fabricated in supersaturated CaP solutions prepared using chemical reagents. In this study, a so-called RKM solution prepared using clinically approved infusion fluids was employed as a supersaturated CaP solution. A D-CaP layer consisting of submicron spherical particles was successfully fabricated on a polystyrene substrate by immersing the substrate in the RKM solution for 24 h. When the immersion period was prolonged from 24 to 72 h, amount of CaP and DNA on the substrate increased. However, the gene transfer capability of the D-CaP layer for the CHO-K1 cells was kept unchanged irrespective of the immersion period. In the RKM solution process, immersion period of 24 h was found to be long enough for gene transfer application of the D-CaP layer. More importantly, the D-CaP layer fabricated by the RKM solution process exhibited a significantly higher gene transfer capability than our previous D-CaP layer fabricated in the conventional CaP solution with the same DNA concentration. The RKM solution process for the fabrication of D-CaP layers was found to be advantageous to the previous process in terms of not only safety but the layers gene transfer capability.

Abstract: Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.

Abstract: Post-Transcriptional Gene Silencing and RNA interference (RNAi) are terms describing the specific suppression of genes by complementary dsRNA. High-efficiency transfection is an essential first step for achieving effective gene knockdown. Non-viral transfection include chemical-transfection and electrotransfection. For a particular cell line, select the appropriate transfection method is very important. In this study, we compared a variety of commercially available reagents and electrotransfection, approaches to define methods for efficient delivery of siRNA to NB4 cells. Fluorescently-labelled siRNA were tested. We report that different methods have different effects. The results provide guidelines for optimal siRNA-mediated knockdown in the NB4 cells.