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Christine Bassila, Rose Ghemrawi, Justine Flayac, D Sean Froese, Matthias R Baumgartner, Jean-Louis Guéant, David Coelho
An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs...
October 19, 2016: Biochimica et Biophysica Acta
Alexandra McCarron, Martin Donnelley, Chantelle McIntyre, David Parsons
Lentiviruses are becoming an increasingly popular choice of gene transfer vehicle for use in the treatment of a variety of genetic and acquired human diseases. As research progresses from basic studies into pre-clinical and clinical phases, there is a growing demand for large volumes of high purity, concentrated vector, and accordingly, the means to produce such quantities. Unlike other viral vectors, lentiviruses are difficult to produce using stable cell lines, therefore transient transfection of adherent cell lines is conventionally used, and this method has proven challenging to up-scale...
October 18, 2016: Journal of Biotechnology
Junyi Che, Anqi Tao, Shun Chen, Xiaoming Li, Yi Zhao, Weien Yuan
Small interfering RNA (siRNA) has increased the hope for highly-efficient treatment of gene-related diseases. However, the stable and efficient delivery of therapeutic nucleic acids is a prerequisite for the successful clinical translation of RNA interfering therapy. To achieve this, we condensed the low molecular weight polyethyleneimine (PEI, Mw < 2000) with 2,6-pyridinedicarboxaldehyde (PDA) to synthesize a biologically responsive and degradable cationic polymer (abbreviated to PDAPEI) which was utilized as a gene vector for the delivery of a VEGF-A shRNA expression plasmid DNA (pDNA)...
October 19, 2016: Scientific Reports
X Chen, Q Lv, Y Liu, W Deng
Although the role of PLCγ2 in apoptotic response has been reported, too little is known about whether PLCγ2 induces liver cell apoptosis during liver regeneration. Therefore, this study firstly packaged Ad-PLCγ2 recombinant adenovirus and primarily evaluated its effect on apoptosis of rat liver cell BRL-3A in vitro. Following ten days of co-transfection of pHBAd-MCMV-GFP-PLCγ2 and pHBAd-BHG into HEK293 cells, viral cytopathic effect (CPE) was apparent. Following three rounds of amplification, tissue culture infectious dose 50 (TCID50) assay showed that the titer value reached 1×1010 PFU/mL...
September 30, 2016: Cellular and Molecular Biology
Igor Slivac, David Guay, Mathias Mangion, Juliette Champeil, Bruno Gaillet
Delivery of nucleic acid-based molecules in human cells is a highly studied approach for the treatment of several disorders including monogenic diseases and cancers. Non-viral vectors for DNA and RNA transfer, although in general less efficient than virus-based systems, are particularly well adapted mostly due to the absence of biosafety concerns. Non-viral methods could be classified in two main groups: physical and vector-assisted delivery systems. Both groups comprise several different methods, none of them universally applicable...
October 14, 2016: Expert Opinion on Biological Therapy
Ramón Román, Joan Miret, Federica Scalia, Antoni Casablancas, Martí Lecina, Jordi J Cairó
Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters tested...
October 7, 2016: Journal of Biotechnology
Md Nasimuzzaman, Danielle Lynn, Rebecca Ernst, Michele Beuerlein, Richard H Smith, Archana Shrestha, Scott Cross, Kevin Link, Carolyn Lutzko, Diana Nordling, David W Russell, Andre Larochelle, Punam Malik, Johannes C M Van der Loo
Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34(+) cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy...
2016: Molecular Therapy. Methods & Clinical Development
Somayeh Kadkhodayan, Azam Bolhassani, Seyed Mehdi Sadat, Shiva Irani, Fatemeh Fotouhi
Cell penetrating peptides (CPPs) or protein transduction domains (PTDs) have been known as a new field in cargo delivery. These peptides such as Tat, Pep-1 and Cady-2 are able to deliver genes and biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, the efficiency of pEGFP-N1 eukaryotic vector for expression of HIV-1 Tat-Nef fusion was evaluated in HEK-293T cells using TurboFect transfection reagent. In addition, the recombinant GST-Tat-Nef protein was generated in E...
October 6, 2016: Current Drug Delivery
Julie Ry Gustafsson, Georgia Katsioudi, Shohreh Issazadeh-Navikas, Birgitte Rahbek Kornum
BACKGROUND: Efficient and specific knockdown of proteins in post-mitotic cells such as differentiated neurons can be difficult to achieve. Further, special care must be taken to maintain the health of neurons in vitro. We wanted to achieve knockdown in primary cerebellar granule neurons, which can be effectively grown in Neurobasal™ media. NEW METHOD: We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer...
October 4, 2016: Journal of Neuroscience Methods
Megan C Schwarz, Marion Sourisseau, Michael M Espino, Essanna S Gray, Matthew T Chambers, Domenico Tortorella, Matthew J Evans
The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site...
September 2016: MSphere
Moran Golan, Valeria Feinshtein, Ayelet David
The key for successful gene silencing is to design a safe and efficient siRNA delivery system for the transfer of therapeutic nucleic acids into the target cells. Here, we describe the design of hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer displaying multiple copies of octaarginine (R8) and its use in promoting the effective delivery of small interfering RNA (siRNA) molecules intracellularly. Fluorescein-5-isothiocyanate (FITC)-labeled HPMA copolymer-bound R8 (P-R8-FITC) was synthesized with increasing R8 molar ratios (4 to 9...
October 1, 2016: European Journal of Pharmaceutics and Biopharmaceutics
Mariette Andersson, Helle Turesson, Alessandro Nicolia, Ann-Sofie Fält, Mathias Samuelsson, Per Hofvander
Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines...
October 3, 2016: Plant Cell Reports
Xijiao Guo, Ling Feng, Jing Jia, Ruibao Chen, Jun Yu
INTRODUCTION: Preeclampsia is a severe pregnancy complication mostly due to inadequate vascular dilation and remodeling of spiral arteries. VEGF, the major factor for angiogenesis, is necessary for modulating angiogenic processes in the placenta. Hence reduction of VEGF in gestational hypertension may also lead to hypoperfusion and subsequent hypoxia of the fetus in hypertensive pregnancy. METHODS: This study aimed at elucidating the mechanism of action of VEGF in preeclampsia...
October 2016: Placenta
Cheng-Qiong Luo, Yoonjeong Jang, Lei Xing, Peng-Fei Cui, Jian-Bin Qiao, Ah Young Lee, Hyeon-Jeong Kim, Myung-Haing Cho, Hu-Lin Jiang
Lung cancer has been a leading cause of cancer mortality worldwide and aerosol-mediated gene therapy endows numerous advantages compared to other traditional modalities. Here, we reported a folic acid (FA)-modified hyperbranched polyspermine (HPSPE) with prominent biocompatibility for lung cancer cell targeted gene therapy. FA was decorated to the HPSPE via an amidation reaction and the physicochemical properties of nanoplexes formed with DNA were characterized. Gel electrophoresis study elucidated that the designed polymer was capable to condense DNA and protect it from degradation by DNase I...
September 25, 2016: International Journal of Pharmaceutics
Jared L Spidel, Benjamin Vaessen, Yin Yin Chan, Luigi Grasso, J Bradford Kline
Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential...
September 24, 2016: Journal of Immunological Methods
Li Lei, Li Tang
Schwann cells (SCs) have been reported as a possible source of neurotrophic support for spiral ganglion neurons (SGNs). This study was aimed to investigate whether S100A4 was contributed in the functional effects of SCs on SGNs. SCs were transfected with S100A4 vector or small interfering RNA (siRNA) against S100A4, and the transfection efficiency was verified by quantitative PCR (qPCR) and Western blot. The migration of transfected SCs was determined by Transwell assay, and the expression levels of vascular endothelial growth factor precursor (VEGF) and matrix metallopeptidase 9 (MMP-9) were measured by Western blot...
September 26, 2016: Bioengineered
Dabing Xue, Ying Liang, Siliang Duan, Jian He, Jing Su, Jianmeng Zhu, Nan Hu, Jianming Liu, Yongxiang Zhao, Xiaoling Lu
Whole tumor cell vaccines have shown much promise, but demonstrated poor efficiency in phase III trials. In this study, we modified MDA-MB‑231 tumor cells (MDA-MB‑231Gal+) to express α-1, 3-galactosyltransferase (α-1, 3-GT) protein, to potentially enhance antitumor effect of whole tumor cell vaccines. MDA-MB‑231 tumor cell vaccines were transfected with a reconstructed lentiviral containing α-1, 3-GT genes. Tumor growth, tumorigenesis and survival of Hu-NOD-SCID mice were observed when tumor-bearing mice were injected with tumor cell vaccines...
September 23, 2016: Oncology Reports
Xin Liu, Yun-Pan Li, Zhen-Min Zhong, Hui-Qi Tan, Hao-Peng Lin, Shao-Jun Chen, Yu-Cai Fu, Wen-Can Xu, Chi-Ju Wei
The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes...
September 24, 2016: Applied Biochemistry and Biotechnology
Lipeng Ren, Wei Han, Hong Yang, Fen Sun, Shuanshuan Xu, Shuxian Hu, Mingzhi Zhang, Xin He, Jinlian Hua, Sha Peng
Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity...
October 21, 2016: Biochemical and Biophysical Research Communications
Yun-Ui Bae, Jae-Wan Huh, Bieong-Kil Kim, Hyeon Young Park, Young-Bae Seu, Kyung-Oh Doh
Cholesterol is an important cell membrane component and has been used as co-lipid for cationic liposome to enhance gene delivery. However, the role of cholesterol in transfection efficiency has not been fully understood. In this study, transfection efficiency of liposome was measured after cholesterol was added to the cell culture medium. As a result, addition of cholesterol increased transfection efficiency of several liposomes consisting of different lipid components in various cells (AGS, CHO, COS7 and, MCF7)...
September 21, 2016: Biochimica et Biophysica Acta
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