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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) 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,

(2) 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. It was found that formulations with DNA: CM-β-CD (Carboxymethyl-beta-cyclodextrin): Pluronic-F127 1:3:3 and 1:3 DNA: CM-β-CD are the most stable formulations indicating high incorporation of DNA within CM-β -CD. Gel electrophoresis revealed DNA with low CM-β -CD concentration has formed a more stable complex. Samples 1:3 DNA: CM-β-CD and 1:3:3 DNA: CM-β-CD: Pluronic-127 show no DNA fragment suggesting good condensation of DNA inside cyclodextrin cavity. This was confirmed by fluorescence data where fluorescence intensity was reduced for samples DNA: CM-β-CD 1:3. Overall the findings showed that Carboxymethyl-beta-cyclodextrin (as a novel non-viral gene vector) was able to provide condensation and protection to the DNA, with and without Pluronic-F127, at low concentration. pDNA/CM-β-CD complex has not just shown to be able to transfect COS 7 and SH-SY5Y cell lines but it gave a higher transfection efficiency than that produced by the TransIT-LT1 commercial transfection reagent.

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

(3) 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,

(4) 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,

(5) Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects.[TOP]

Pubmed ID :29185158
Publication Date : //
In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan-dextran (CS-DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS-DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS-DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS-DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.

Authors : Perumal Vanathi, Arfuso Frank, Chen Yan, Fox Simon, Dharmarajan Arun M,

(6) Gene delivery ability of polyethylenimine and polyethylene glycol dual-functionalized nanographene oxide in 11 different cell lines.[TOP]

Pubmed ID :29134085
Publication Date : //
We recently developed a polyethylenimine (PEI) and polyethylene glycol (PEG) dual-functionalized reduced graphene oxide (GO) (PEG-nrGO-PEI, RGPP) for high-efficient gene delivery in HepG2 and Hela cell lines. To evaluate the feasibility and applicability of RGPP as a gene delivery carrier, we here assessed the transfection efficiency of RGPP on gene plasmids and siRNA in 11 different cell lines. Commercial polyalkyleneimine cation transfection reagent (TR) was used as comparison. In HepG2 cells, RGPP exhibited much stronger delivery ability for siRNA and large size plasmids than TR. For green fluorescent protein (GFP) plasmid, RGPP showed about 47.1% of transfection efficiency in primary rabbit articular chondrocytes, and about 27% of transfection efficiency in both SH-SY5Y and A549 cell lines. RGPP exhibited about 37.2% of GFP plasmid transfection efficiency in EMT6 cells and about 26.0% of GFP plasmid transfection efficiency in LO2 cells, but induced about 33% of cytotoxicity in both cell lines. In 4T1 and H9C2 cell lines, RGPP had less than 10% of GFP plasmid transfection efficiency. Collectively, RGPP is a potential nano-carrier for high-efficiency gene delivery, and needs to be further optimized for different cell lines.

Authors : Wu Liping, Xie Jinshan, Li Tan, Mai Zihao, Wang Lu, Wang Xiaoping, Chen Tongsheng,

(7) Trans-acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high-throughput microbioreactor system.[TOP]

Pubmed ID :29023997
Publication Date : //
A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

Authors : Hsu Chih-Jung, Jain Harsh V, Williams Abasha, Wang Julie, Lute Scott C, Beaucage Serge L, Brorson Kurt A,

(8) Lipid-Coated Gold Nanoparticles Functionalized by Folic Acid as Gene Vectors for Targeted Gene Delivery in vitro and in vivo.[TOP]

Pubmed ID :28967206
Publication Date : //
Lipid-based nanoparticles as gene vectors have attracted considerable attention for their high gene transfection efficiency and low cytotoxicity. In our previous work, we synthesized gold nanoparticles/dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) (GDD) as anionic lipid- and pH-sensitive gene vectors. To further realize targeted gene transfection, a series of gold nanoparticles/DODAB/DOPE/DOPE-folic acid (DOPE-FA) with various ratios of DOPE-FA were prepared and termed as GFn (for which n=1.0, 2.5, 5.0, 7.5, or 10.0 %). The gene transfection efficiency mediated by GF2.5 can reach about 85 % for MCF-7 (FA-receptor-positive cells), higher than those of the negative control (GDD, 35 %) and positive control (Lipofectamine 2000, 65 %). However, GF2.5 does not further promote gene transfection into A549 (FA-receptor-negative cells). The higher gene transfection efficiency for MCF-7 cells can be attributed to enhanced cellular uptake efficiency mediated by the FA targeting ability. Furthermore, GF2.5 was also found to accumulate at the specific tumor site and showed enhanced in vivo gene delivery ability. In addition, no significant harm was observed for the main tissues of the mice after treatment with GF2.5. Therefore, GF2.5, with the targeting ability and improved transfection efficiency, shows promise for its utility in gene therapy for tumor cells that overexpress FA receptors. We believe the results of this study will find more broad applications in gene therapy.

Authors : Du Baoji, Gu Xiaoxiao, Han Xu, Ding Guanyu, Wang Yuling, Li Dan, Wang Erkang, Wang Jin,

(9) High Efficiency Low Cost Fibroblast Nucleofection for GMP Compatible Cell-based Gene Therapy.[TOP]

Pubmed ID :28824316
Publication Date : //
Dermal fibroblast is a powerful tool for the study of DNA delivery in development of both cell therapy and tissue engineering products. Using genetic modification, fibroblasts can be diversely adapted and made suitable for clinical gene therapy. In this study, we first compared several non-viral transfection methods including nucleofection in rat and human primary dermal fibroblast. In addition, the original protocol for nucleofection of primary mammalian fibroblasts was modified in order to achieve the highest possible transfection efficiency, as determined by flow cytometry analysis of the green fluorescent protein (GFP) expression. : the results showed that transfection performance of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) yielded the best transfection efficiency with rat dermal fibroblasts and ITS (insulin, transferrin, and sodium selenite solution) was comparable to the standard nucleofection solution for human dermal fibroblasts. Our results suggest a promising application of the modified nucleofection method for GMP compatible therapeutic translational medical research.

Authors : Zhang Ziyang, Slobodianski Alex, Arnold Astrid, Nehlsen Jessica, Hopfner Ursula, Schilling Arndt F, Perisic Tatjana, Machens Hans-Günther,

(10) Dual Recombinase-Mediated Cassette Exchange by Tyrosine Site-Specific Recombinases.[TOP]

Pubmed ID :28815493
Publication Date : //
Recombinase-mediated cassette exchange, or RMCE, is a genome engineering tool that can be used to swap DNA fragments of interest between two DNA molecules. In a variation of RMCE, called dual RMCE, the exchange of DNA fragments is mediated by two recombinases in contrast to one recombinase in the classic RMCE reaction. Under optimal conditions, the efficiency of dual RMCE can be quite high: up to ~45% of the transfected cells depending on the recombinase pair used to mediate the replacement reaction. Here we describe protocols for preparing for, performing, and optimizing the parameters of dual RMCE.

Authors : Voziyanova Eugenia, Anderson Rachelle P, Voziyanov Yuri,