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RNotion Transfection Reagent _1 ml

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[#2201500] RNotion Transfection Reagent _1 ml

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(1) Enhanced Intercellular Delivery of cRGD-siRNA Conjugates by an Additional Oligospermine Modification.[TOP]

Pubmed ID :30087937
Publication Date : //
Small interfering RNA (siRNA), consisting a 21-mer duplex molecule, is often modified by conjugation with specific ligands to enhance its capacity for tissue-specific delivery. However, these attempts are hampered by the low permeability of negatively charged RNA molecules to enter the cell membrane. In this study, we designed and synthesized siRNA conjugates modified with cationic oligospermine and cyclic RGD (cRGD) to overcome the low-membrane permeability of siRNA. The siRNA conjugate, which contains 15 spermines and a cRGD peptide, showed sufficient gene-silencing activity at 250 nM final concentration without a transfection reagent. Under these conditions, the cationic oligospermine and cRGD-siRNA conjugate did not show any cytotoxicity.

Authors : Nakamoto Kosuke, Akao Yukihiro, Furuichi Yasuhiro, Ueno Yoshihito,

(2) Antibody-mediated oral delivery of therapeutic DNA for type 2 diabetes mellitus.[TOP]

Pubmed ID :30065848
Publication Date : //
Diabetes mellitus (DM) is a chronic progressive metabolic disease that involves uncontrolled elevation of blood glucose levels. Among various therapeutic approaches, GLP-1 prevents type 2 diabetes mellitus (T2DM) patients from experiencing hyperglycemic episodes. However, the short half-life (< 5 min) and rapid clearance of GLP-1 often limits its therapeutic use. Here, we developed an oral GLP-1 gene delivery system to achieve an extended antidiabetic effect.

Authors : Cha Seungbin, Lee Sun Hwa, Kang Sung Hun, Hasan Mohammad Nazmul, Kim Young Jun, Cho Sungpil, Lee Yong-Kyu,

(3) Polysaccharide-modified nanoparticles with intelligent CD44 receptor targeting ability for gene delivery.[TOP]

Pubmed ID :30022822
Publication Date : //
Hyaluronic acid (HA) and chondroitin sulfate (CD) are endogenous polysaccharides. In recent years, they have aroused the interest of scientists because of specific binding to CD44 receptors, which are overexpressed in several types of tumors.

Authors : Lin Wen Jen, Lee Wei Chi,

(4) Influence of YKL-40 gene RNA interference on the biological behaviors of endometrial cancer HEC-1A cells.[TOP]

Pubmed ID :30008865
Publication Date : //
The present study aimed to investigate the effects of chitinase-3-like protein 1 (YKL-40) gene RNA interference on the biological behaviors and enhanced chemosensitivity of endometrial cancer (EC) HEC-1A cells. YKL-40 small interfering (si)RNA was transduced into EC HEC-1A cells using a lentivirus. The experiment was divided into three groups: The experimental group was transfected with YKL-40 siRNA (si-YKL-40); the mock-treatment group was transfected with transfection reagent only; and the blank control group was left untreated. A reverse transcription-quantitative polymerase chain reaction was performed to investigate the mRNA expression levels of YKL-40. The biological behaviors, including cell proliferation, migration, invasion and apoptosis, were detected by MTT and Transwell assays, and flow cytometry (FCM) analysis, respectively. The results of the present study demonstrated that the mRNA expression levels of YKL-40 were downregulated within HEC-1A cells upon transfection with si-YKL-40 (P<0.05). The proliferative, migratory and invasive abilities of HEC-1A cells were inhibited by si-YKL-40 (P<0.05). The mRNA expression levels of YKL-40 were upregulated within HEC-1A cells following treatment with cisplatin (P<0.05). FCM analysis revealed that the average cellular apoptosis rate increased following the inhibition of YKL-40 gene expression via siRNA (P<0.05). Therefore, the YKL-40 gene may be associated with the proliferative, migratory, invasive and anti-apoptotic ability of HEC-1A cells. YKL-40 downregulation may enhance the sensitivity of human EC HEC-1A cells to chemotherapy.

Authors : Li Lili, Fan Jiangtao, Li Dahai, Liu Yan, Shrestha Poonam, Zhong Chunyan, Xia Xiuhong, Huang Xiaobing,

(5) Early growth response factor-1 DNA enzyme 1 inhibits the formation of abdominal aortic aneurysm in rats.[TOP]

Pubmed ID :29977360
Publication Date : //
The aim of the present study was to characterize the effects of early growth response factor-1 DNA enzyme (EDRz) in a rat abdominal aortic aneurysm (AAA) model to determine the mechanism by which EDRz inhibits AAA and affects the formation of AAA by regulating the activity of matrix metalloproteinase (MMP)-2 and MMP-9. EDRz was transfected into the abdominal aorta of rats using the jetPRIME transfection reagent following infusion with elastase. Fluorescent microscopy, hematoxylin and eosin staining, ultrastructural analysis, reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemical analysis were performed to characterize the response to EDRz. The EDRz group showed minimal aneurysm formation when compared with the control group, with significantly lower aortic diameter expansion (2.5±0.1 vs. 3.5±0.1 mm; P<0.05). Early growth response factor 1 (Egr-1) mRNA and protein levels were significantly decreased in the EDRz group, as expected. The decrease in Egr-1 was accompanied by decreases in the mRNA and protein levels of MMP-2 and MMP-9 (P<0.05). Transfection of the Egr-1 specific synthetic DNA enzyme EDRz significantly reduced AAA following elastase infusion in rats, at least in part due to the decreased expression of downstream MMP-2 and MMP-9.

Authors : Wang Shi, Dong Haipeng, Liu Chengwei, Xu Guichao, Hu Xinhua, Fan Yichuan, Chen Liting,

(6) Synthesis, Characterization, and Function of an RNA-Based Transfection Reagent.[TOP]

Pubmed ID :29927123
Publication Date : //
A synthetic 8-mer, amphipathic, trans-acting poly-2'-O-methyluridylic thiophosphate triester RNA element (2'-OMeUtaPS) can be prepared using solid-phase synthesis protocols. 2'-OMeUtaPS efficiently mediates the delivery of uncharged polyA-tailed phosphorodiamidate morpholino (PMO) sequences in HeLa pLuc 705 cells, as evidenced by flow cytometry measurements. In this cell line, 2'-OMeUtaPS-mediated transfection of an antisense polyA-tailed PMO sequence induces alternative splicing of an aberrant luciferase pre-mRNA splice site, leading to restoration of functional luciferase, as quantitatively measured using a typical luciferase assay. 2'-OMeUtaPS is also potent at delivering an uncharged antisense polyA-tailed PMO sequence in muscle cells of the mdx mouse model of muscular dystrophy; targeting the polyA-tailed PMO sequence against a splice site of the pre-mRNA encoding mutated dystrophin triggers an alternate splicing event that results in excision of the mutated exon (exon 23) from the pre-mRNA and production of functional dystrophin, as demonstrated by agarose gel electrophoresis. © 2018 by John Wiley & Sons, Inc.

Authors : Jain Harsh V, Boehler Jessica F, Nagaraju Kanneboyina, Beaucage Serge L,

(7) Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells.[TOP]

Pubmed ID :29886030
Publication Date : //
For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/l for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change.

Authors : Stuible Matthew, Burlacu Alina, Perret Sylvie, Brochu Denis, Paul-Roc Béatrice, Baardsnes Jason, Loignon Martin, Grazzini Eric, Durocher Yves,

(8) Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin.[TOP]

Pubmed ID :29858073
Publication Date : //
In recent years, synthetic mRNA-based applications to produce desired exogenous proteins in cells have been gaining importance. However, systemic delivery of synthetic mRNA can result in unspecific uptake into undesired cells or organs and, thereby, fail to target desired cells. Thus, local and targeted delivery of synthetic mRNA becomes increasingly important to reach the desired cell types and tissues. In this study, intradermal delivery of synthetic mRNA using a hollow microneedle injection-based method was evaluated. Furthermore, an ex vivo porcine skin model was established to analyze synthetic mRNA-mediated protein expression in the skin following intradermal delivery. Using this model, highly efficient delivery of synthetic mRNA was demonstrated, which resulted in detection of high levels of secretable humanized Gaussia luciferase (hGLuc) protein encoded by the microinjected synthetic mRNA. Interestingly, synthetic mRNA injected without transfection reagent was also able to enter the cells and resulted in protein expression. The established ex vivo porcine skin model can be used to evaluate the successful production of desired proteins after intradermal delivery of synthetic mRNAs before starting with in vivo experiments. Furthermore, the use of microneedles enables patient-friendly, painless, and efficient delivery of synthetic mRNAs into the dermis; thus, this method could be applied for local treatment of different skin diseases as well as for vaccination and immunotherapy.

Authors : Golombek Sonia, Pilz Martin, Steinle Heidrun, Kochba Efrat, Levin Yotam, Lunter Dominique, Schlensak Christian, Wendel Hans Peter, Avci-Adali Meltem,

(9) Novel carbamate-linked quaternary ammonium lipids containing unsaturated hydrophobic chains for gene delivery.[TOP]

Pubmed ID :29801966
Publication Date : //
In this paper, two novel carbamate-linked quaternary ammonium lipids (MU18: a lipid with a mono-ammonium head; GU18: a lipid with a Gemini-ammonium head) containing unsaturated hydrophobic chains were designed and synthesized. The chemical structures of the synthetic lipids were characterized by infrared spectrum, ESI-MS, H NMR, C NMR, and HPLC. For investigating the effect of unsaturation on gene delivery, the previous reported saturated cationic liposomes (MS18 and GS18) were used as comparison. Cationic liposomes were prepared by using these cationic lipids and neutral lipid DOPE at the molar ratio of 1:1. Particle sizes and zeta potentials of the cationic liposomes were studied to show that they were suitable for gene transfection. The binding abilities of the cationic liposomes were investigated by gel electrophoresis at various N/P ratios from 0.5/1 to 8/1. The results indicated that the binding ability of GU18 was much better than MU18 and the saturated cationic liposomes (MS18 and GS18). DNA transfection of these liposomes comparable to commercially available reagent (DOTAP) was achieved in vitro against Hela, HepG-2 and NCI-H460 cell lines. GU18 showed higher transfection at the N/P ratio of 3/1 than other cationic liposomes and the positive control, DOTAP. All of the liposomes presented a relatively low cytotoxicity, which was measured by MTT. Therefore, the synthetic lipids bearing unsaturated hydrophobic chains and Gemini-head could be promising candidates for gene delivery.

Authors : Zhou Hengjun, Yang Jian, Du Yanyan, Fu Shuang, Song Chenxi, Zhi Defu, Zhao Yinan, Chen Huiying, Zhang Shubiao, Zhang Shufen,

(10) [Research of enhanced green fluorescent protein gene transfer with ultrasound-mediated microbubble destruction in bone defects].[TOP]

Pubmed ID :29798609
Publication Date : //
To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction.

Authors : Li Shiwei, Xie Xiaoli, Yang Xiaodong, Liu Lijun, Tang Xueyang,