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Min Liang, Tingting Peng, Yawei Shi
Periaxin, a protein of noncompact myelin, is specifically expressed in the peripheral nervous system (PNS). There are two protein isoform L-periaxin and S-Periaxin by alternative splicing of periaxin gene, playing an important role in the initiation of myelin formation. So far, 18 different mutation sites in L-periaxin gene have been found to induce the peripheral demyelinating neurological charcot-marie-tooth diseases subtype 4F (CMT4F). The technique of activation of transcription activator-like effector nucleases (TALENS) was used to knock out the L-periaxin gene in RSC 96 cell line of Rattus...
December 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Morten Lundh, Kaja Pluciñska, Marie S Isidor, Patricia S S Petersen, Brice Emanuelli
OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations...
October 2017: Molecular Metabolism
Trisha M Westerhof, Wesley A Cox-Muranami, Guann-Pyng Li, Mark Bachman, Hung Fan, Edward L Nelson
Advancing knowledge of biological mechanisms has come to depend upon genetic manipulation of cells and organisms, relying upon cellular cloning methods that remain unchanged for decades, are labor and time intensive, often taking many months to come to fruition. Thus, there is a pressing need for more efficient processes. We have adapted a newly developed micropallet array platform, termed the "ferro-core micropallet array", to dramatically improve and accelerate the process of isolating clonal populations of adherent cells from heterogeneous mixtures retaining the flexibility of employing a wide range of cytometric parameters for identifying colonies and cells of interest...
October 12, 2017: Scientific Reports
Chen-Lin Feng, Yan-Xing Han, Hui-Hui Guo, Xiao-Lei Ma, Zhi-Qiang Wang, Lu-Lu Wang, Wen-Sheng Zheng, Jian-Dong Jiang
Our previous work proved that sequence specific double strand RNA (dsRNA-p21) effectively activated p21 gene expression of colorectal cancer (CRC) cells and consequently suppressed CRC growth. However, efficient delivery system is a significant challenge to achieve sufficient therapy. In this study, a self-assembled HA/PEI/dsRNA-p21 ternary complex (TC-dsRNA-p21) was developed for the tumor-target delivery of dsRNA-p21 into CRC cells. Hyaluronic acid (HA) was introduced to shield the PEI/dsRNA-p21 binary complexes (BC-dsRNA-p21) for reducing the cytotoxicity of PEI and for increasing the tumor-targeted intracellular uptake by cancer cells through HA-CD44 mediated endocytosis...
November 2017: Drug Delivery
Nan Jiang, Qian Chen, Sheng Cao, Bo Hu, Yi-Jia Wang, Qing Zhou, Rui-Qiang Guo
The development of an efficient delivery system is critical for the successful treatment of cardiovascular diseases using non‑viral gene therapies. Cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the transfection of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes may improve gene therapy. The present study used a novel strategy involving ultrasound‑targeted microbubbles and peptide nucleic acid (PNA)‑binding nuclear localization signals (NLS)...
October 2, 2017: Molecular Medicine Reports
Qian Han, Liang Li, Hengpo Liang, Yaqiong Li, Jiacun Xie, Zhibin Wang
BACKGROUND LncRNA X inactive specific transcript (XIST) was reported to function as an oncogene in nasopharyngeal carcinoma cells (NPC) by sponging miR-34a-5p. However, the role of XIST in modulating the radiosensitivity of NPC cells and its mechanism still remain undefined. MATERIAL AND METHODS The expressions of XIST and miR-29c in NPC cells were evaluated by qRT-PCR. CNE1 and CNE2 cells were transfected with si-XIST, pcDNA-XIST, miR-29c mimics, anti-miR-29c, or respective controls by Lipofectamine 2000. The effects of XIST knockdown and miR-29c overexpression on cell proliferation, survival fraction, and γ-H2AX expression were investigated by CCK-8 assay, colony formation assay, immunofluorescence, and Western blot, respectively...
October 6, 2017: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
Laure Gibot, Muriel Golzio, Marie-Pierre Rols
Cell membranes can be transiently permeabilized under the application of electric pulses. This process, called electropermeabilization or electroporation, allows hydrophilic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. The method is nowadays used in clinics to treat cancers. Vaccination and gene therapy are other fields of application of DNA electrotransfer. A description of the mechanisms can be assayed by using different complementary systems with increasing complexities (models of membranes, cells cultivated in 2D and 3D culture named spheroids, and tissues in living mice) and different microscopy tools to visualize the processes from single molecules to entire animals...
2017: Advances in Anatomy, Embryology, and Cell Biology
Pei-Ge Chen, Yan-Jing Guan, Guang-Ming Zha, Xian-Qin Jiao, He-Shui Zhu, Cheng-Yu Zhang, Yue-Ying Wang, He-Ping Li
To explore the role of IRF3/IRF7 during inflammatory responses, we investigated the effects of swine IRF3/IRF7 on TLR4 signaling pathway and inflammatory factors expression in porcine kidney epithelial PK15 cell lines. We successfully constructed eukaryotic vectors PB-IRF3 and PB-IRF7, transfected these vectors into PK15 cells and observed GFP under a fluorescence microscope. In addition, RT-PCR was also used to detect transfection efficiency. We found that IRF3/IRF7 was efficiently overexpressed in PK15 cells...
September 22, 2017: Oncotarget
X Gao, X Li, C Qian, F Li, Y Zhang, L Dang, X Xiao, F Liu, H Li, X Zhang
MicroRNA involves in regulating behavior of neural stem/precursor cells (NSPCs), thus it offers the potential to treat central nervous system disease. However, the effect of miR-21 on NSPCs remains unknown. In this study, we demonstrated that miR-21 reduced proliferation and promoted neural differentiation in NSPCs via regulating the activation of AKT and GSK-3β signaling pathways in vitro. During differentiation of NSPCs, the expression of miR-21 was increased in a time-dependent manner by qRT-PCR. Synthesized pre-miR-21 or anti-miR-21 was transfected into NSPCs, thereby efficiently overexpressing or knocking down miR-21...
October 31, 2016: Cellular and Molecular Biology
A Karlitepe, H Kabadayi, S Vatansever, M Gurdal, C Gunduz, G Ercan
AIM: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). METHODS: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RT-PCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR...
September 2017: Experimental Oncology
Jun Yu, Jing Jia, Xijiao Guo, Ruibao Chen, Ling Feng
INTRODUCTION: Excessive circulating sFlt1 plays a major role in the pathogenesis of preeclampsia (PE). Using RNAi to silence sFlt1 may be a therapy for treating PE. Because of the rapid degradation of siRNA, gene therapy in vivo remains limited. Poly-amidoamine (PAMAM) has been demonstrated to be an excellent nanocarrier for siRNA delivery with no discernible toxicity. METHODS: The aim of the present study was to investigate the therapeutic effect of siRNA-sFlt1-PAMAM on PE...
October 2017: Placenta
Phan Nguyen Nhi Nguyen, Kong Bung Choo, Chiu-Jung Huang, Shigeki Sugii, Soon Keng Cheong, Tunku Kamarul
BACKGROUND: Introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to 'reprogram' somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs)...
September 29, 2017: Stem Cell Research & Therapy
Jingying Hou, Lei Wang, Huibao Long, Hao Wu, Quanhua Wu, Tingting Zhong, Xuxiang Chen, Changqing Zhou, Tianzhu Guo, Tong Wang
BACKGROUND: Cardiac stem cells (CSCs) transplantation has been regarded as an optimal therapeutic approach for cardiovascular disease. However, inferior survival and low differentiation efficiency of these cells in the local infarct site reduce their therapeutic efficacy. In this study, we investigated the influence of hypoxia preconditioning (HP) on CSCs survival and cardiogenic differentiation in vitro and explored the relevant mechanism. METHODS: CSCs were obtained from Sprague-Dawley rats and cells of the third passage were cultured in vitro and exposed to hypoxia (1% O2)...
September 29, 2017: Stem Cell Research & Therapy
Ying Xiong, Liqian Liu, Ying Qiu, Lili Liu
Melanoma is one of the most aggressive malignant tumors, with increasing incidence, poor prognosis, and lack of any effective targeted therapy. Abnormal expression of miR-29a has been found in several types of cancers, including melanoma. However, the role of miR-29a in melanoma, and the molecular mechanism by which miR-29a represses melanoma are rarely reported. Cell transfection efficiency was determined by Quantitative real-time PCR or Western blot. The cell viability, apoptosis, migration, and invasion were respectively determined by Cell Counting Kit-8 assay, PI/FITC-Annexin V staining assay, wound healing assay and Transwell assay...
September 26, 2017: Oncology Research
Qingfen Zheng, Li Bai, Sujun Zheng, Mei Liu, Jinyan Zhang, Ting Wang, Zhongwei Xu, Yu Chen, Jiansheng Li, Zhongping Duan
Current therapeutic strategies cannot eradicate hepatitis B virus covalently closed circular DNA (HBV cccDNA), which accounts for the persistence of HBV infection. Very recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‑associated protein 9 (Cas9) system has been used as an efficient and powerful tool for viral genome editing. Given that the primary duck hepatocyte (PDH) infected with duck hepatitis B virus (DHBV) has been widely used to study human HBV infection in vitro, the present study aimed to demonstrate the targeted inhibition of DHBV DNA, especially cccDNA, by the CRISPR/Cas9 system using this model...
September 19, 2017: Molecular Medicine Reports
Jianlin Long, Yuping Yang, Tianyi Kang, Wei Zhao, Hao Cheng, Yujiao Wu, Ting Du, Beibei Liu, Yang Li, Feng Luo, Maling Gou
Nanoparticles have great promise for gene delivery. However, the transfection efficiency of nanoparticle-based gene delivery systems is always unsatisfied to meet the requirement of effective gene therapy. Herein, we used low dosage paclitaxel to enhance a nanoscaled gene delivery system that was self-assembled from DOTAP and MPEG-PLA(DPP), creating a paclitaxel-encapsulated DPP nanoparticle (P-DPP). The encapsulated low-dosage paclitaxel significantly improved the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells, in some of which the transfection efficiency is as high as 92%...
September 25, 2017: ACS Applied Materials & Interfaces
R Eggers, A Philippi, M O Altmeyer, F Breinig, M J Schmitt
Immune cells become increasingly attractive as delivery system for immunotoxins in cancer therapy in order to reduce the intrinsic toxicity and severe side effects of chimeric protein toxins. In this study, we investigated the potential of human primary T cells to deliver a secreted immunotoxin through transient mRNA transfection. The chimeric protein toxin was directed towards the neovasculature of cancer cells by fusing a truncated version of Pseudomonas exotoxin A (PE38) to human vascular endothelial growth factor (VEGF) and to the single chain variable fragment (scFv) of anti-Her2/neu...
September 22, 2017: Gene Therapy
Xiaotian T Fang, Dag Sehlin, Lars Lannfelt, Stina Syvänen, Greta Hultqvist
BACKGROUND: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.. METHODS: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost...
2017: Biological Procedures Online
Li Zhang, Qing Zhou, Wen Song, Kaimin Wu, Yumei Zhang, Yimin Zhao
Surface functionalization by small interfering RNA (siRNA) is a novel strategy for improved implant osseointegration. A gene delivery system with safety and high transfection activity is a crucial factor for an siRNA-functionalized implant to exert its biological function. To this end, polyethylene glycol (PEG) and polyethylenimine (PEI) dual-functionalized graphene oxide (GO; nGO-PEG-PEI) may present a promising siRNA vector. In this study, nanosized nGO-PEG-PEI was prepared and optimized for siRNA delivery...
October 11, 2017: ACS Applied Materials & Interfaces
Jaitip Tipanee, Thierry VandenDriessche, Marinee K Chuah
Transposons have emerged as promising vectors for gene therapy that can potentially overcome some of the limitations of commonly used viral vectors. Transposons stably integrate into the target cell genome, enabling persistent expression of therapeutic genes. Transposons have evolved from being used as basic tools in biomedical research to bona fide therapeutics. Currently, the most promising transposons for gene therapy applications are derived from Sleeping Beauty (SB) or piggyBac (PB). Stable transposition requires co-delivery of the transposon DNA with the corresponding transposase gene, mRNA, or protein...
August 22, 2017: Human Gene Therapy
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