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DNotion Transfection Reagent _ 4 x 1.2ml

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[#2201410] DNotion Transfection Reagent _ 4 x 1.2ml


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(1) [Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells].[TOP]

Pubmed ID :30644257
Publication Date : //
To discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells.

Authors : Yan Liang, Jiang Jin, Ma Chongwen, Li Rui, Xia Yayi,

(2) Gene delivery into Siberian sturgeon cell lines by commercial transfection reagents.[TOP]

Pubmed ID :30644030
Publication Date : //
The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1. Then, the complexes were used to transfect three Siberian sturgeon cell lines derived from the heart, head kidney, and gonad. Cytotoxicity and transfection efficiency were measured via flow cytometry after propidium iodide staining. No significant cytotoxicity was observed at the optimal treatment conditions in all cases, with the exception of Lipofectamine 2000-treated gonad-derived cells. Although the transfection efficiencies in A. baerii cells were generally low, X-tremeGENE HP DNA Transfection Reagent showed the highest transfection efficiency at ratios of 2:1 or 4:1, depending on the cell type. Hence, X-tremeGENE HP DNA Transfection Reagent can be used to effectively transfer foreign genes into three A. baerii cell lines.

Authors : Lee Ji Hun, Lee Seung Tae, Nam Yoon Kwon, Gong Seung Pyo,

(3) Insights into siRNA transfection in suspension: Efficient gene silencing in human mesenchymal stem cells encapsulated in hyaluronic acid hydrogel.[TOP]

Pubmed ID :30642167
Publication Date : //
Small interfering RNAs (siRNAs) are powerful tools for post-transcriptional gene silencing which offers enormous opportunities for tissue engineering applications. However, poor serum stability, inefficient intracellular delivery, and inevitable toxicity of transfection reagents are the key barriers for their clinical translation. Thus innovative strategies that allow safe and efficient intracellular delivery of the nucleic acid drugs at the desired site is urgently needed for a smooth clinical translation of therapeutically appealing siRNA-based technology. In this regard, we have developed an innovative siRNA transfection protocol that employs a short incubation time of just 5-minutes. This allows easy transfection in suspension followed by transplantation of the cells in a hyaluronic acid (HA) hydrogel system. We also report here the unique ability of siRNA to bind HA that was quantified by siRNA release and rheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA in suspension transfection conditions within 30-minutes using native HA, although removal of excess HA by centrifugation seem to be essential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with or without removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removal of excess reagent after 30 minutes. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improved knockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) as determined by PrestoBlueTM assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the protein levels by Western blot analysis. We believe this novel transfection method could be applied for in vivo applications as it allows minimal manipulation of cells that are to be transplanted and reduce toxicity.

Authors : Paidikondala Maruthibabu, Nawale Ganesh N, Varghese Oommen P,

(4) Establishment and characterization of an immortalized renal cell line of the Chinese tree shrew (Tupaia belangeri chinesis).[TOP]

Pubmed ID :30637496
Publication Date : //
The Chinese tree shrew holds a great potential as a viable animal model in biomedical research, especially for infectious diseases and neuropsychiatric disorders. A thorough understanding of the innate immunity, which represents the first line that defends the host against viral infection, of the Chinese tree shrew, is needed. However, the progress is hindered by the lack of a proper cell line for research usage. In this study, we established a cell line that is applicable to the study of tree shrew innate immune responses against viral infections. The Chinese tree shrew primary renal cells (TSPRCs) were immortalized by simian virus 40 large T antigen (SV40LT) transduction, and the immortalized cells were termed TSR6 (tree shrew renal cell #6). TSR6 showed a similar morphology to TSPRCs and expressed the epithelial cell-specific marker cytokeratin 18 (KRT18). In addition, TSR6 could be transfected by transfection reagent and was suitable for CRISPR/Cas9-mediated gene editing. Infection of Newcastle disease virus (NDV) or herpes simplex virus 1 (HSV-1) in TSR6 induced the mRNA expression of tree shrew interferon-β (tIFNB1) and myxovirus resistance protein 1 (tMx1) in a dose- and time-dependent manner. Collectively, we successfully established a tree shrew renal cell line and demonstrated that this cell line was suitable for the study of the innate immune response to viral infections.

Authors : Gu Tianle, Yu Dandan, Li Yu, Xu Ling, Yao Yu-Lin, Yao Yong-Gang,

(5) Engineered histidine-enriched facial lipopeptides for enhanced intracellular delivery of functional siRNA to triple negative breast cancer cells.[TOP]

Pubmed ID :30628773
Publication Date : //
Cytosolic delivery of functional siRNA remains the major challenge to develop siRNA based therapeutics. Designing clinically safe and effective siRNA transporter to deliver functional siRNA across the plasma and endosomal membrane remains a key hurdle. With the aim of improving endosomal release, we have designed cyclic and linear peptide based transporters having Arg-DHis-Arg template. Computational studies show that Arg-DHis-Arg template is also stabilized by Arg-His side-chain hydrogen bonding interaction at physiological pH, which dissociates at lower pH. The overall atomistic interactions were examined by molecular dynamics simulations, which indicate that extent of peptide-siRNA assembly formation depends greatly on physicochemical properties of the peptides. Our designed peptides having Arg-DHis-Arg template and two lipidic moieties facilitate high yield of intracellular delivery of siRNA. Additionally, unsaturated lipid, linoleic acid moieties were introduced to promote fusogenicity and facilitate endosomal release and cytosolic delivery. Interestingly, such protease-resistant peptides provide serum stability to siRNA and exhibit high efficacy of erk1 and erk2 gene silencing in triple negative breast cancer cell line. The peptide having two linoleyl moieties demonstrated comparable efficacy with commercial transfection reagent HiPerFect, as evidenced by erk1 and erk2 gene knockdown experiment. Additionally, our study shows that ERK1/2 silencing siRNA and doxorubicin loaded gramicidin mediated combination therapy is more effective than siRNA mediated gene silencing based monotherapy for "Triple negative" breast cancer (TNBC) treatment.

Authors : Biswas Abhijit, Chakraborty Kasturee, Dutta Chiranjit, Mukherjee Sanchita, Gayen Paramita, Jan Somnath, Mallick Argha Mario, Bhattacharyya Dhananjay, Sinha Roy Rituparna,

(6) Raman tweezers microspectroscopy of circa 100 nm extracellular vesicles.[TOP]

Pubmed ID :30620023
Publication Date : //
The technique of Raman tweezers microspectroscopy (RTM) for the global biomolecular content characterization of a single extracellular vesicle (EV) or a small number of EVs or other nanoscale bioparticles in an aqueous dispersion in the difficult-to-access size range of near 100 nm is described in detail. The particularities and potential of RTM are demonstrated using the examples of DOPC liposomes, exosomes from human urine and rat hepatocytes, and a mixed sample of the transfection reagent FuGENE in diluted DNA solution. The approach of biomolecular component analysis for the estimation of the main biomolecular contributions (proteins, lipids, nucleic acids, carotenoids, etc.) is proposed and discussed. Direct Raman evidence for strong intra-sample biomolecular heterogeneity of individual optically trapped EVs, due to variable contributions from nucleic acids and carotenoids in some preparations, is reported. On the basis of the results obtained, we are making an attempt to convince the scientific community that RTM is a promising method of single-EV research; to our knowledge, it is the only technique available at the moment that provides unique information about the global biomolecular composition of a single vesicle or a small number of vesicles, thus being capable of unravelling the high diversity of EV subpopulations, which is one of the most significant urgent challenges to overcome. Possible RTM applications include, among others, searching for DNA biomarkers, cancer diagnosis, and discrimination between different subpopulations of EVs, lipid bodies, protein aggregates and viruses.

Authors : Kruglik Sergei G, Royo Félix, Guigner Jean-Michel, Palomo Laura, Seksek Olivier, Turpin Pierre-Yves, Tatischeff Irène, Falcón-Pérez Juan M,

(7) Bone marrow mesenchymal stem cells: improving transgene expression level, transfection efficiency and cell viability.[TOP]

Pubmed ID :30610819
Publication Date : //
Advanced cancer is a catastrophic medical condition that is generally treated with surgery and conventional anticancer drugs, which are very toxic and often fail. A promising alternative is using genetically engineered mesenchymal stem cells. A popular method for genetically engineering mesenchymal stem cells (MSCs) is by employing transfection reagents. Nevertheless, a serious limitation of this procedure is its consistently low transfection efficiency. Therefore, the utility of transfection reagents in regenerative medicine - including cancer treatment - might increase strikingly by increasing their transfection efficiency and maintaining, to the greatest extent possible, cell viability and transgene expression levels. The purpose of this study was to analyze various effects on gene expression level, transfection efficiency, and cell viability by increasing the volume of transfection reagents and the plasmid DNA mass.

Authors : Gonzalez Villarreal Carlos, Said Fernandez Salvador, Soto Dominguez Adolfo, Padilla Rivas Gerardo, Garza Treviño Elsa, Rodriguez Rocha Humberto, Martinez Rodriguez Herminia,

(8) A Neutralizing Aptamer to TGFBR2 and miR-145 Antagonism Rescue Cigarette Smoke- and TGF-β-Mediated CFTR Expression.[TOP]

Pubmed ID :30595527
Publication Date : //
Transforming growth factor β (TGF-β), signaling induced by cigarette smoke (CS), plays an important role in the progression of airway diseases, like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD), and in smokers. Chronic bronchitis is characterized by reduced mucociliary clearance (MCC). Cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in normal MCC. TGF-β and CS (via TGF-β) promote acquired CFTR dysfunction by suppressing CFTR biogenesis and function. Understanding the mechanism by which CS promotes CFTR dysfunction can identify therapeutic leads to reverse CFTR suppression and rescue MCC. TGF-β alters the microRNAome of primary human bronchial epithelium. TGF-β and CS upregulate miR-145-5p expression to suppress CFTR and the CFTR modifier, SLC26A9. miR-145-5p upregulation with a concomitant CFTR and SLC26A9 suppression was validated in CS-exposed mouse models. While miR-145-5p antagonism rescued the effects of TGF-β in bronchial epithelial cells following transfection, an aptamer to block TGF-β signaling rescues CS- and TGF-β-mediated suppression of CFTR biogenesis and function in the absence of any transfection reagent. These results demonstrate that miR-145-5p plays a significant role in acquired CFTR dysfunction by CS, and they validate a clinically feasible strategy for delivery by inhalation to locally modulate TGF-β signaling in the airway and rescue CFTR biogenesis and function.

Authors : Dutta Rajib K, Chinnapaiyan Srinivasan, Rasmussen Lawrence, Raju S Vamsee, Unwalla Hoshang J,

(9) Enhanced gene delivery in tumor cells using chemical carriers and mechanical loadings.[TOP]

Pubmed ID :30592721
Publication Date : //
Intracellular delivery of DNA is considered a challenge in biological research and treatment of diseases. The previously reported transfection rate by commercially available transfection reagents in cancer cell lines, such as the mouse lung tumor cell line (TC-1), is very low. The purpose of this study is to introduce and optimize an efficient gene transfection method by mechanical approaches. The combinatory transfection effect of mechanical treatments and conventional chemical carriers is also investigated on a formerly reported hard-to-transfect cell line (TC-1). To study the effect of mechanical loadings on transfection rate, TC-1 tumor cells are subjected to uniaxial cyclic stretch, equiaxial cyclic stretch, and shear stress. The TurboFect transfection reagent is exerted for chemical transfection purposes. The pEGFP-N1 vector encoding the green fluorescent protein (GFP) expression is utilized to determine gene delivery into the cells. The results show a significant DNA delivery rate (by ~30%) in mechanically transfected cells compared to the samples that were transfected with chemical carriers. Moreover, the simultaneous treatment of TC-1 tumor cells with chemical carriers and mechanical loadings significantly increases the gene transfection rate up to ~ 63% after 24 h post-transfection. Our results suggest that the simultaneous use of mechanical loading and chemical reagent can be a promising approach in delivering cargoes into cells with low transfection potentials and lead to efficient cancer treatments.

Authors : Hadi Amin, Rastgoo Abbas, Haghighipour Nooshin, Bolhassani Azam, Asgari Fatemeh, Soleymani Sepehr,

(10) Developing a novel cholesterol-based nanocarrier with high transfection efficiency and serum compatibility for gene therapy.[TOP]

Pubmed ID :30579664
Publication Date : //
Primary cells are sensitive to culture conditions, which can be more difficult to get efficient transfection. The purpose of this study is to develop a serum-compatible cholesterol-based nanocarrier for delivering therapeutic nucleic acids into cells efficiently for future clinical gene therapy.

Authors : Chang Karen, Chang Fu-Hsiung, Chen Min-Huey,