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increase transfection efficiency

Ikramy A Khalil, Seigo Kimura, Yusuke Sato, Hideyoshi Harashima
We report on the development of a highly efficient gene delivery system based on synergism between octaarginine (R8), a representative cell penetrating peptide, and YSK05, a recently developed pH-sensitive cationic lipid. Attaching a high density of R8 on the surface of YSK05 nanoparticles (NPs) that contained encapsulated plasmid DNA resulted in the formation of positively charged NPs with improved transfection efficiency. To avoid the development of a net positive charge, we controlled the density and topology of the R8 peptide through the use of a two-step coating methodology, in which the inner lipid coat was modified with a low density of R8 which was then covered with an outer neutral YSK05 lipid layer...
February 13, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
Ching-Wei Tsai, Ze-Wei Lin, Wen-Fang Chang, Yi-Fan Chen, Wei-Wen Hu
Indolicidin (IL) is a cationic antimicrobial peptide and our previous study has demonstrated its potential as a cell penetrating peptide (CPP) to promote gene delivery. However, the cytotoxicity of IL arisen from its membrane perturbation capacity may restrict its clinical application. To promote gene delivery safety and efficiency, an almost mirror-symmetric IL derivative, SAP10 (RRWKFFPWRR-CONH 2 ), was designed in this study. All-atom molecular dynamics (MD) simulations were performed to understand the association between SAP10 and model lipid bilayers...
February 5, 2018: Colloids and Surfaces. B, Biointerfaces
Zhiying Wang, He Shen, Saijie Song, Liming Zhang, Wei Chen, Jianwu Dai, Zhijun Zhang
Delivery of functional genes into stem cells shows great application prospect in DNA-based tissue engineering. However, comparing with epithelial cells and cancer cells, stem cells usually exhibit low gene transfection efficiency. To enhance the transfection efficiency, non-viral gene delivery in combination with biomaterial scaffolds, has raised increasing interests from researchers in tissue engineering. Nanofibers fabricated by electrospinning technique mimicking extracellular matrix (ECM) are widely used in tissue engineering applications...
April 1, 2018: Journal of Nanoscience and Nanotechnology
Dan-Hua Xu, Xiao-Yin Wang, Yan-Long Jia, Tian-Yun Wang, Zheng-Wei Tian, Xin Feng, Yin-Na Zhang
Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF-1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells...
February 14, 2018: Journal of Cellular and Molecular Medicine
Shi-Lung Lin, Donald C Chang, Shao-Yao Ying
Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo...
2018: Methods in Molecular Biology
C X Ma, G Y Zheng
Objective: To construct lentiviral-mediated EphA2 overexpression vectors, transfect them into human lens epithelial cells (HLE-B3) in vitro, and investigate the effect of EphA2 gene overexpression on the proliferation and apoptosis of HLE-B3 exposed to high-concentration dexamethasone. Methods: Experimental Study. The pCDH-CMV- MCS-EF1-RFP plasmid was set up by the digestion of NOTⅠand XbaⅠ double restriction enzyme and ligation of CE ligase, and then the plasmid was transformed into DH10B cells. Seven clons were picked for enzymatic digestion and the clons with correct results were chosen for sequencing...
February 11, 2018: [Zhonghua Yan Ke za Zhi] Chinese Journal of Ophthalmology
Tae-Jin Lee, Min Suk Shim, Taekyung Yu, Kyunghee Choi, Dong-Ik Kim, Soo-Hong Lee, Suk Ho Bhang
Adipose-derived stem cells (ADSCs) have the potential to treat ischemic diseases. In general, ADSCs facilitate angiogenesis by secreting various pro-angiogenic growth factors. However, transplanted ADSCs have a low therapeutic efficacy in ischemic tissues due to their poor engraftment and low viability. Stromal cell-derived factor-1α (SDF-1α) improves the survival rate of stem cells transplanted into ischemic regions. In this study, we developed acid-degradable poly(ethylene glycol)-poly(amino ketal) (PEG-PAK)-based micelles for efficient intracellular delivery of SDF-1α plasmid DNA...
February 9, 2018: International Journal of Molecular Sciences
J Pei, P Li, Z-Y Zhang, H-L Zhang, Y-H Gao, D-Y Wang, Y Zheng, X Xu, J-Z Cui
OBJECTIVE: To explore the role of transgelin-2 TAGLN2 in the development and progression of meningioma and the potential regulatory. MATERIALS AND METHODS: TAGLN2 knockdown expression and overexpression in vitro models were constructed using lentivirus in meningioma cell line CH157; their corresponding transfection efficiencies were verified by qRT-PCR and Western Blot. Actions of TAGLN2 on the proliferation of meningioma cells were explored by CCK8 and colony formation assays...
January 2018: European Review for Medical and Pharmacological Sciences
Jamie L Everman, Cydney Rios, Max A Seibold
The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs)...
2018: Methods in Molecular Biology
Xiaojuan Cui, Ying Sun, Ming Shen, Keqi Song, Xia Yin, Wen Di, Yourong Duan
Chemotherapy-induced activation of cell survival pathways leads to drug resistance. MicroRNAs post-transcriptionally regulate gene expression in many biological pathways. Paclitaxel (PTX) is one of the first-line chemotherapy drugs for ovarian cancer and induces the activation of the EGFR/ERK pathway that leads to tumor cell proliferation, survival, invasion and drug resistance. MicroRNA-7 (miR-7) has the ability to suppress the EGFR/ERK pathway. To sensitize chemotherapy, we developed mPEG-PLGA-PLL nanoparticles for the simultaneous co-delivery of PTX and miR-7...
February 7, 2018: ACS Applied Materials & Interfaces
Valentina Pomatto, Erika Cottone, Paolo Cocci, Matteo Mozzicafreddo, Gilberto Mosconi, Erik Russel Nelson, Francesco Alessandro Palermo, Patrizia Bovolin
Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01-25 μM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate)...
February 2, 2018: Journal of Steroid Biochemistry and Molecular Biology
C Prezioso, D Scribano, D M Rodio, C Ambrosi, M Trancassini, A T Palamara, V Pietropaolo
John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle...
February 5, 2018: Virology Journal
Wei-Jiang Liu, Xue Li, Yuan-Lin Liu, Peng Wang, Yue Fan, Tian-Yi Teng, Bo-Qian Bai, Yue Tong, Wei Zhang, Yi Zhang
OBJECTIVE: To investigate the effect of MicroRNA-3963(miR-3963) on the adipogenic differentiation of mouse bone-derived mesenchymal stem cells(MSC). METHODS: MSCs were isolated from C57BL/6 mice bone fragment and transfected with miR-3963 mimic, miR-3963 inhibitor and negative control. The expression of miR-3963 and transfection efficiency were detected by q-PCR. These transfected cells were induced to adipocytes and stained with oil red O after 14 days culture...
February 2018: Zhongguo Shi Yan Xue Ye Xue za Zhi
Matjaž Stenovec, Saša Trkov Bobnar, Tina Smolič, Marko Kreft, Vladimir Parpura, Robert Zorec
AIM: Alzheimer disease (AD) is largely considered a neuron-derived insult, but also involves failure of astroglia. A recent study indicated that mutated presenilin 1 (PS1M146V), a putative endoplasmic reticulum (ER) Ca2+ channel with decreased Ca2+ conductance, impairs the traffic of astroglial peptidergic vesicles. Whether other pathogenically relevant PS1 mutants, such as PS1ΔE9, which code for ER channel with putative increased Ca2+ conductance, similarly affect vesicle traffic, is unknown...
February 2, 2018: Acta Physiologica
Q Guo, G Mintier, M Ma-Edmonds, D Storton, X Wang, X Xiao, B Kienzle, D Zhao, John N Feder
Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR')...
February 1, 2018: Scientific Reports
Zhang Zhang, Shunfang Qiu, Xiaopeng Zhang, Wei Chen
BACKGROUND: Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is theoretically safer, because it does not promote transgene integration into the host genome. Additionally, the simplicity and speed of the procedure increases the attractiveness of electroporation...
January 30, 2018: BMC Biotechnology
Xiuying Li, Peiyuan Kang, Zhuo Chen, Sneha Lal, Li Zhang, Jeremiah J Gassensmith, Zhenpeng Qin
Efficient delivery to the cell nucleus remains a significant challenge for many biomolecules, including anticancer drugs, proteins and DNAs. Despite numerous attempts to improve nuclear import including the use of nuclear localization signal (NLS) peptides and nanoparticle carriers, they are limited by the nanoparticle size, conjugation method, dependence on the functional nuclear import and intracellular trafficking mechanisms. To overcome these limitations, here we report that the nanomechanical force from plasmonic nanobubbles increases nuclear membrane permeability and promotes universal uptake of macromolecules into the nucleus, including macromolecules that are larger than the nuclear pore complex and would otherwise not enter the nucleus...
January 29, 2018: Chemical Communications: Chem Comm
Wen Meng, Shi-Chong Han, Cui-Cui Li, Hui-Jun Dong, Xiao-Jia Wang
To ensure efficient virus replication, herpes simplex virus type 1 (HSV-1) encodes several viral proteins to counter host defense response upon infection. Among these proteins, the multifunctional viral protein γ34.5 crucially interferes with or disrupts several antiviral pathways at multiple levels. The current study shows that γ34.5 utilizes nucleolar protein NOP53 to facilitate the dephosphorylation of eukaryotic initiation factor eIF2α for efficient viral translation. Our study shows that: (1) ectopic expression of NOP53 greatly increases the intracellular and extracellular viral yields of HSV-1 (wild strain F) in type I interferon-deficient Vero cells, and more subtly promotes viral replication of γ34...
January 24, 2018: Cell Death & Disease
Zhaojun Song, Yongjie Ye, Zhi Zhang, Jieliang Shen, Zhenming Hu, Zhigang Wang, Jiazhuang Zheng
Various gene delivery systems have been widely studied for the acute spinal cord injury (SCI) treatment. In the present study, a novel type of brain-derived neurotrophic factor (BDNF)-loaded cationic nanobubbles (CNBs) conjugated with MAP-2 antibody (mAbMAP-2/BDNF/CNBs) was prepared to provide low-intensity focused ultrasound (LIFU)-targeted gene therapy. In vitro experiments, the ultrasound-targeted tranfection to BDNF overexpressioin in neurons and efficiently inhibition neuronal apoptosis have been demonstrated, and the elaborately designed mAbMAP-2/BDNF/CNBs can specifically target to the neurons...
January 19, 2018: Biochemical and Biophysical Research Communications
I Fernandez-Piñeiro, A Pensado, I Badiola, A Sanchez
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...
January 17, 2018: European Journal of Pharmaceutics and Biopharmaceutics
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