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High Efficiency Transfection Reagent

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[#GT1211-01] High Efficiency Transfection Reagent


GT1211-01 | High Efficiency Transfection Reagent , 0.5 ml.
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(1) High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology.[TOP]

Pubmed ID :30290128
Publication Date : //
The Labcyte Echo acoustic liquid handler allows accurate droplet ejection at high speed from a source well plate to a destination plate. It has already been used in various miniaturized biological assays, such as quantitative PCR (q-PCR), quantitative real-time PCR (q-RT-PCR), protein crystallization, drug screening, cell dispensing, and siRNA transfection. However, no plasmid DNA transfection assay has been published so far using this dispensing technology. In this study, we evaluated the ability of the Echo 550 device to perform plasmid DNA transfection in 384-well plates. Due to the high throughput of this device, we simultaneously optimized the three main parameters of a transfection process: dilution of the transfection reagent, DNA amount, and starting DNA concentration. We defined a four-step protocol whose optimal settings allowed us to transfect HeLa cells with up to 90% efficiency and reach a co-expression of nearly 100% within transfected cells in co-transfection experiments. This fast, reliable, and automated protocol opens new ways to easily and rapidly identify optimal transfection settings for a given cell type. Furthermore, it permits easy software-based transfection control and multiplexing of plasmids distributed on wells of a source plate. This new development could lead to new array applications, such as human ORFeome protein expression or CRISPR-Cas9-based gene function validation in nonpooled screening strategies.

Authors : Colin Béatrice, Deprez Benoit, Couturier Cyril,

(2) Optimization of the Linker Length of Mannose-Cholesterol Conjugates for Enhanced mRNA Delivery to Dendritic Cells by Liposomes.[TOP]

Pubmed ID :30233368
Publication Date : //
Liposomes (LPs) as commonly used mRNA delivery systems remain to be rationally designed and optimized to ameliorate the antigen expression of mRNA vaccine in dendritic cells (DCs). In this study, we synthesized mannose-cholesterol conjugates (MP-CHs) by click reaction using different PEG units (PEG, PEG, and PEG) as linker molecules. MP-CHs were fully characterized and subsequently used to prepare DC-targeting liposomes (MP-LPs) by a thin-film dispersion method. MP-LPs loaded with mRNA (MP-LPX) were finally prepared by a simple self-assembly method. MP-LPX displayed bigger diameter (about 135 nm) and lower zeta potential (about 40 mV) compared to MP-LPs. The transfection experiment on DC2.4 cells demonstrated that the PEG length of mannose derivatives had significant effect on the expression of GFP-encoding mRNA. MP-LPX containing MP-CH can achieve the highest transfection efficiency (52.09 ± 4.85%), which was significantly superior to the commercial transfection reagent Lipo 3K (11.47 ± 2.31%). The optimal DC-targeting MP-LPX showed an average size of 132.93 ± 4.93 nm and zeta potential of 37.93 ± 2.95 mV with nearly spherical shape. Moreover, MP-LPX can protect mRNA against degradation in serum with high efficacy. The uptake study indicated that MP-LPX enhanced mRNA expression mainly through the over-expressing mannose receptor (CD206) on the surface of DCs. In conclusion, mannose modified LPs might be a potential DC-targeting delivery system for mRNA vaccine after rational design and deserve further study on the delivery profile and anti-tumor efficacy.

Authors : Wang Fazhan, Xiao Wen, Elbahnasawy Mostafa A, Bao Xingting, Zheng Qian, Gong Linhui, Zhou Yang, Yang Shuping, Fang Aiping, Farag Mohamed M S, Wu Jinhui, Song Xiangrong,

(3) Photoluminescent Cationic Carbon Dots as efficient Non-Viral Delivery of Plasmid SOX9 and Chondrogenesis of Fibroblasts.[TOP]

Pubmed ID :29728593
Publication Date : //
With the increasing demand for higher gene carrier performance, a multifunctional vector could immensely simplify gene delivery for disease treatment; nevertheless, the current non- viral vectors lack self-tracking ability. Here, a type of novel, dual-functional cationic carbon dots (CDs), produced through one-step, microwave-assisted pyrolysis of arginine and glucose, have been utilized as both a self-imaging agent and a non-viral gene vector for chondrogenesis from fibroblasts. The cationic CDs could condense the model gene plasmid SOX9 (pSOX9) to form ultra-small (10-30 nm) nanoparticles which possessed several favorable properties, including high solubility, tunable fluorescence, high yield, low cytotoxicity and outstanding biocompatibility. The MTT assay indicated that CDs/pSOX9 nanoparticles had little cytotoxicity against mouse embryonic fibroblasts (MEFs) compared to Lipofectamine2000 and PEI (25 kDa). Importantly, the CDs/pSOX9 nanoparticles with tunable fluorescence not only enabled the intracellular tracking of the nanoparticles, but also could successfully deliver the pSOX9 into MEFs with significantly high efficiency. Furthermore, the CDs/pSOX9 nanoparticles-mediated transfection of MEFs showed obvious chondrogenic differentiation. Altogether, these findings demonstrated that the CDs prepared in this study could serve as a paradigmatic example of the dual-functional reagent for both self-imaging and effective non-viral gene delivery.

Authors : Cao Xia, Wang Jianping, Deng Wenwen, Chen Jingjing, Wang Yan, Zhou Jie, Du Pan, Xu Wenqian, Wang Qiang, Wang Qilong, Yu Qingtong, Spector Myron, Yu Jiangnan, Xu Ximing,

(4) Cholic acid-modified polyethylenimine: in vitro and in vivo studies.[TOP]

Pubmed ID :29593402
Publication Date : //
Low-molecular-weight polyethylenimine has lower cytotoxicity than high molecular weight polyethylenimine, but it is not an efficient transfection agent because of limitations of DNA delivery into the cytoplasm. Therefore, in the present study, the hydrophobic modification of low-molecular-weight polyethylenimine (PEI 2 kDa [PEI2]) by cholic acid (ChA) was performed to form PEI2-ChA, and in vitro and in vivo studies were performed. Results indicate that the nanoplexes of PEI2-ChA with gWIZ-GFP have greater transfection efficiency (27%) in NT8e cell lines as evaluated by flow cytometry and also observed by fluorescence imaging. The present study concluded that the transferrin-containing nanoplexes of PEI2-ChA conjugates with plasmid p53 warrant clinical trials in humans after exhaustive animal studies for use as a novel gene delivery system.

Authors : Dube Brahmanand, Pandey Abhijeet, Joshi Ganesh, Mulherkar Rita, Sawant Krutika,

(5) Production of Transgenic Mice Through Sperm-Mediated Gene Transfer Using Magnetic Nano-Carriers.[TOP]

Pubmed ID :29490755
Publication Date : //
Current methods of transgenic animal production suffer from low efficiency, cumbersome operation, and high cost. Magnetic nanoparticles (MagNPs) have several characteristics, such as a high carrying efficiency, non-immunogenicity, and strong targeting inducible via magnetic fields, that make them well-suited for use in the generation of transgenic animals. In this study, we used magnetic nano-carriers combined with sperm-mediated gene transfer (SMGT) to generate transgenic mice that harbor the enhanced green fluorescent protein (EGFP) gene. Exogenous plasmid DNA loaded onto Fe3O4 MagNPs were first delivered into mouse sperm cells under a magnetic field. Transfected sperm cells were then incubated with oocytes to complete fertilization, and transgenic mice were successfully generated though embryo transplantation. We demonstrate that this method is exceedingly facile, fast, and cost-effective, with higher transfection efficiency than that of conventional liposome methods.

Authors : Wang Yan, Zhao Xiang, Du Wei, Liu Jinghao, Chen Wenjie, Sun Changjiao, Cui Bo, Zeng Zhanghua, Shen Yue, Gao Fei, Wang Anqi, Liu Guoqiang, Cui Haixin,

(6) Insights into the Influences of Carboxymethyl-β-Cyclodextrin on DNA Formulations Characteristics and Gene Transfection Efficiency.[TOP]

Pubmed ID :29484997
Publication Date : //
Gene therapy is an expanding field and it can treat genetic and acquired diseases.

Authors : Elsana Hassan, Mysina Svetlana, Elkordy Eman Ali, Carr-Wilkinson Jane, Elkordy Amal Ali,

(7) Functionalized Asymmetric Bola-Type Amphiphiles for Efficient Gene and Drug Delivery.[TOP]

Pubmed ID :29462991
Publication Date : //
The studies of bolaamphiphile-based nanoparticles as delivery vectors are still rudimentary and under development. In this study, several asymmetric bolaamphiphiles containing lysine and another moiety with special functions, such as pH-sensitive or cell-targeting property, were designed and synthesized. The potentials of these bolaamphiphile-based nanoparticles as versatile vectors for both nucleic acids and chemical drugs were studied. With the presence of 1,2-dioleoyl--glycero-3-phosphoethanolamine (DOPE), these amphiphiles could be prepared into bolasomes, which showed good DNA binding ability and could condense plasmid DNA into nanoparticles with appropriate size and surface potential. , which has a pH-sensitive histidine on one head, exhibited higher transfection efficiency than the symmetric counterpart and comparable efficiency to commercially available transfection reagent. Mechanism studies confirmed that the bolaplexes formed from might induce the highest cellular uptake and the best endosomal escape ability. On the other hand, these bolaamphiphiles also exhibited good drug loading ability. The self-assembly vesicles could efficiently encapsulate the hydrophobic anti-cancer drug doxorubicin (DOX) in aqueous solution with high drug loading content and encapsulation efficiency. Confocal laser scanning microscopy (CLSM) experiment and cell viability assay exhibited a controlled release of the drug with the assistance of bolasomes. It was shown that such bolaamphiphiles have great potential as nano-vectors for both drug and gene or their co-delivery.

Authors : Huang Zheng, Zhao Dong-Mei, Deng Xuan, Zhang Ji, Zhang Yi-Mei, Yu Xiao-Qi,

(8) Characterization of DNA Condensation by Conformationally Restricted Dipeptides and Gene Delivery.[TOP]

Pubmed ID :29372986
Publication Date : //
A wide variety of non-viral vectors have been developed for gene delivery in past few decades but find limited applications mainly due to lower encapsulation, endosomal entrapment, high toxicity and low transfection efficiency. In this work, we explored plasmid DNA binding ability of several low molecular weight dipeptides containing α,β-dehydrophenylalanine (ΔF) and found that an arginine containing dipeptide, arginine-α,β-dehydrophenylalanine (R-ΔF) condensed pEGFP-N1 plasmid into positively charged spherical nanoparticles of size 250–275 nm. Single molecule techniques showed that R-ΔF interacted with the plasmid DNA in a dose dependent manner which was accompanied by a decrease in diffusion time of the plasmid DNA as well as release of the bound fluorophore. The plasmid DNA in R-ΔF-plasmid complex (R-ΔF-Pl) was stable against DNase action. A pH dependent release of the plasmid DNA from R-ΔF-Pl was observed and the released plasmid DNA retained its natural conformation at endosomal pH, as evidenced from time correlated single photon counting. R-ΔF-Pl was biocompatible and showed ready uptake in HEK 293T cells. Transfection assays using reporter plasmids for green fluorescent protein (GFP), luciferase enzyme and chloramphenicol acetyltransferase (CAT) showed R-ΔF mediated gene delivery both in the presence and absence of serum in the medium. Ease of synthesis, homogenous assembly and biocompatibility balanced with significant expression of gene of interest make R-ΔF a potential vector for development for in vivo application.

Authors : Khatri Anjali, Mishra Aseem, Chauhan Virander Singh,

(9) Development and characterisation of chondroitin sulfate- and hyaluronic acid-incorporated sorbitan ester nanoparticles as gene delivery systems.[TOP]

Pubmed ID :29355685
Publication Date : //
Glycosaminoglycans (GAGs) are natural polymers that are broadly used in gene delivery systems to increase stability as well as decrease toxicity and nonspecific interactions, thereby increasing transfection efficiency. In this work, we propose sorbitan ester-based lipid nanoparticles (SENS) functionalised with the GAGs chondroitin sulfate (CS) and hyaluronic acid (HA) as gene delivery systems. For this purpose, we describe the design and evaluation of these nanosystems loaded with plasmid DNA, including an evaluation of their physicochemical characteristics, stability properties, ability to protect and efficiently transfect cells with Enhanced Green Fluorescent Protein plasmid (pEGFP) in vitro, and biocompatibility both in vitro and in vivo. We confirm that molecules with high biological value and targeting potential, such as HA and CS, can be successfully incorporated into our recently developed sorbitan ester-based nanoparticles (SENS) and that this incorporation leads to effective stabilisation of both nanosystems as well as protects plasmid DNA. We demonstrated that the aforementioned incorporation of HA and CS enables long-term stability of the nanosystems in both liquid and lyophilised states, which is a remarkable property that can aid in their transfer to industry. The ability of these functionalised nanosystems to transfect the A549 cell line without compromising cell viability was also shown, as well as their innocuous safety profile in vivo. Thus, we provide valuable evidence of the suitable properties and potential of these hybrid nanoparticles as gene delivery systems.

Authors : Fernandez-Piñeiro I, Pensado A, Badiola I, Sanchez A,

(10) Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork.[TOP]

Pubmed ID :29340644
Publication Date : //
To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS).

Authors : Aktas Zeynep, Rao Hongyu, Slauson Sarah R, Gabelt B'Ann T, Larsen Inna V, Sheridan Rachael T C, Herrnberger Leonie, Tamm Ernst R, Kaufman Paul L, Brandt Curtis R,