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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) Application of ion pair chromatography coupled with mass spectrometry to assess antisense oligonucleotides concentrations in living cells.[TOP]

Pubmed ID :30462105
Publication Date : //
Antisense oligonucleotides (ASOs) are synthetic bioactive compounds used as therapeutic agents in clinical trials. They act by binding to complementary sequences of the targeted nucleic acids in cells. Assessing the efficiency of ASO delivery to cells or tissues and the stability of these compounds in different biological systems is important. To answer these questions, we developed a new, quick and reliable method to determine the concentrations of different types of ASOs in treated cells. Ultra-high performance liquid chromatography coupled with mass spectrometry was used for the first time for the separation and determination of the studied compounds in total RNA extracts. To develop a method with the highest possible sensitivity, a central composite design was used to comprehensively optimize the MS parameters. Moreover, the effects of the type and concentration of the ion pair reagent on sensitivity were also examined. Finally, a mobile phase containing methanol, hexafluoroisopropanol and N,N-dimethylbutylamine was selected. The optimized method allowed good linearity, accuracy, precision and sensitivity of ASO detection. Next, these compounds were delivered into cells via transfection at a concentration of 25 nM or 125 nM in 1 mL of cell culture medium. After 48 hours, total RNA was isolated from the treated cells and analyzed with the use of the newly developed method. For the cells treated with a higher concentration of ASO composed of phosphorothioate 2'-O-methyl RNA units, the concentration in solution was 0.96 ± 0.06 μM, while in the case of shorter ASO composed of locked nucleic acid units, it was 0.72 ± 0.06 μM in the total RNA extract.

Authors : Studzińska Sylwia, Cywoniuk Piotr, Sobczak Krzysztof,

(2) Enhanced uptake of plasmid at boronic acid decorated linear polyethylenimines results in higher transfection efficiency.[TOP]

Pubmed ID :30458622
Publication Date : //
High molecular weight polyethylenimines (PEIs) are considered as gold standard for transfection studies; however, cytotoxicity associated with branched ones and lower charge density on linear PEIs as well as lower uptake of the resulting deoxyribonucleic acid (DNA) complexes have limited their applications in clinical studies. In order to address these concerns and improve the uptake efficiency of the DNA complexes of linear polyethylenimine (25 kDa), the polymer was grafted with variable amounts of butylboronic acid to obtain a small series of linear polyethylenimine-butylboronic acid polymers. These modified polymers were allowed to interact with plasmid DNA and the resulting complexes were characterized by physicochemical techniques. Dynamic light scattering data showed the formation of nanosized complexes with positive zeta potential values. Furthermore, when these complexes were evaluated , they not only showed enhanced cell viability but also exhibited higher transfection efficiency as compared to native linear and branched PEIs and a commercially available standard transfection reagent, Lipofectamine 2000.

Authors : Yadav Santosh, Kumar Pradeep,

(3) Highly efficient transfection of human induced pluripotent stem cells using magnetic nanoparticles.[TOP]

Pubmed ID :30323594
Publication Date : //
The delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs).

Authors : Yamoah Megan A, Moshref Maryam, Sharma Janhavi, Chen Wei Chun, Ledford Hannah A, Lee Jeong Han, Chavez Karen S, Wang Wenying, López Javier E, Lieu Deborah K, Sirish Padmini, Zhang Xiao-Dong,

(4) 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,

(5) 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,

(6) Antimicrobial peptide modification enhances the gene delivery and bactericidal efficiency of gold nanoparticles for accelerating diabetic wound healing.[TOP]

Pubmed ID :30187036
Publication Date : //
Impaired angiogenesis and bacterial infection have increasingly been implicated as the major causes of delayed diabetic wound healing. However, there is currently no effective therapy. Here, we optimized a novel gene delivery system based on antimicrobial peptide (LL37) grafted ultra-small gold nanoparticles (AuNPs@LL37, ∼7 nm) for the topical treatment of diabetic wounds with or without bacterial infection. AuNPs@LL37 combines the advantages of cationic AuNPs that condense DNA with those of antibacterial peptides, which are both highly antibacterial and essential for enhancing cellular and nucleus entry to achieve high gene delivery efficiency. AuNPs@LL37 combined with pro-angiogenic (VEGF) plasmids (AuNPs@LL37/pDNAs) significantly improved the gene transfection efficiency in keratinocytes compared with pristine AuNPs/pDNAs, and showed similar expression to Lipo2000/pDNAs (a well-known highly efficient gene transfection agent). Moreover, our therapeutic depot showed higher antibacterial ability than the free antimicrobial peptides and the cationic AuNPs alone in vitro and in vivo due to synergistic effects. Furthermore, the combined system promoted angiogenesis and inhibited bacterial infection in diabetic wounds, resulting in accelerated wound closure rates, faster re-epithelization, improved granulation tissue formation and high VEGF expression. Finally, our therapeutic depot was highly biocompatible in vitro and in vivo, suggesting its potential as a feasible way to treat chronic diabetic wounds.

Authors : Wang Song, Yan Chang, Zhang Ximu, Shi Dezhi, Chi Luxiang, Luo Gaoxing, Deng Jun,

(7) 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,

(8) 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,

(9) 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,

(10) 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,