Using a nanopore for single molecule detection and single cell transfection.

PubMed

Nelson, Edward M; Kurz, Volker; Shim, Jiwook; Timp, Winston; Timp, Gregory

2012-07-07

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

[Recombinant adeno-associated virus mediated RNA interference of angiogenin expression inhibits cell growth of human lung adenocarcinoma].

PubMed

Li, Bai-Ling; Zhang, Guan-Xin; Hou, Xiao-Lei; Tan, Meng-Wei; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Huang, Sheng-Dong

2009-03-01

To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 through adeno-associated virus (AAV)-mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation. Recombinant AAV expressing H1-promoter-induced small-interference- RNA (siRNA) targeting ANG (AAV-shANG) was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and microvessel-density of the carcinoma were observed. In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells (P < 0.01). Cell cycle analysis showed that the proliferation index (PI) of normal A549 cells, AAV-Null cells and AAVshANG cells were 0.32 +/- 0.29, 0.35 +/- 0.38 and 0.31 +/- 0.43, respectively. There was no statistic difference in the PIs among the 3 groups (P > 0.05). In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 +/- 1.5, 9.8 +/- 2.1 and 5.7 +/- 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were (7.7 +/- 3.1)%, (8.5 +/- 5.4)%, (17.1 +/- 8.6)%, respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were (84.8 +/- 9.7)%, (85.8 +/- 9.8)%, and (70.4 +/- 10.1)%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups. AAV-mediated expression of siRNA could reduce the expression of ANG in cancer cells, significantly enough to inhibit cell proliferation, promote cell apoptosis and inhibit tumor growth.

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration.

PubMed

Xu, Kang; Sun, Yanjun; Kh Al-Ani, Mohanad; Wang, Chunli; Sha, Yongqiang; Sung, Kl Paul; Dong, Nianguo; Qiu, Xuefeng; Yang, Li

2018-01-03

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80-90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young's were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Minimally-Invasive Gene Transfection by Chemical and Physical Interaction of Atmospheric Pressure Plasma Flow

NASA Astrophysics Data System (ADS)

Kaneko, Toshiro

2014-10-01

Non-equilibrium atmospheric pressure plasma irradiated to the living-cell is investigated for medical applications such as gene transfection, which is expected to play an important role in molecular biology, gene therapy, and creation of induced pluripotent stem (iPS) cells. However, the conventional gene transfection using the plasma has some problems that the cell viability is low and the genes cannot be transferred into some specific lipid cells, which is attributed to the unknown mechanism of the gene transfection using the plasma. Therefore, the time-controlled atmospheric pressure plasma flow is generated and irradiated to the living-cell suspended solution for clarifying the transfection mechanism toward developing highly-efficient and minimally- invasive gene transfection system. In this experiment, fluorescent dye YOYO-1 is used as the simulated gene and LIVE/DEAD Stain is simultaneously used for cell viability assay. By the fluorescence image, the transfection efficiency is calculated as the ratio of the number of transferred and surviving cells to total cell count. It is clarified that the transfection efficiency is significantly increased by the short-time (<4 sec) and short-distance (<40 mm) plasma irradiation, and the high transfection efficiency of 53% is realized together with the high cell viability (>90%). This result indicates that the physical effects such as the electric field caused by the charged particles arriving at the surface of the cell membrane, and chemical effects associated with plasma-activated products in solution act synergistically to enhance the cell-membrane transport with low-damage. This work was supported by JSPS KAKENHI Grant Number 24108004.

[Role of connective tissue growth factor (CTGF) in proliferation and migration of pancreatic cancer cells].

PubMed

Bai, Yu-chun; Kang, Quan; Luo, Qing; Wu, Dao-qi; Ye, Wei-xia; Lin, Xue-mei; Zhao, Yong

2011-10-01

To explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells. The expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection. CTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells. CTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

Single-cell optoporation and transfection using femtosecond laser and optical tweezers.

PubMed

Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

2013-01-01

In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.

Magnetic iron oxide nanoparticles mediated gene therapy for breast cancer--an in vitro study.

PubMed

Wei, Weizhong; Xu, Chunfang; Wu, Hua

2006-01-01

The aim of this study was to evaluate the feasibility and efficacy of using TRAIL gene to treat breast cancer mediated with a novel carrier - magnetic iron oxide nanoparticles (poly-MAG-1000) coated with PEI. The magnetic iron oxide nanoparticles were used as gene carrier to transfect TRAIL gene into MCF-7 cells. The polyMAG-1000 without TRAIL gene was transfected into the tumor cells as negative control. TRAIL gene transfection with liposome as carrier served as positive control. The apoptosis of cells was detected with TUNEL method. The apoptosis ratio of tumor cells was measured with flow cytometry (FCM). It was found that the apoptosis occurred in the tumor cells after transfection of TRAIL gene mediated by both polyMAG-1000 and liposome. The apoptosis ratio in the group with polyMAG-1000 as gene carrier was (25.11+/-2.85) %, whereas it was (5.06+/- 1.05) % in the control group with polyMAG-1000 (P<0.01). The apoptosis ratio was as low as (18.31+/-2.44) % in the group with liposome as gene carrier (P<0.05, as compared with the group with polyMAG-1000 as gene carrier). It is suggested that TRAIL gene may induce apoptosis in MCF-7 breast cancer cells. The magnetic iron oxide nanoparticles coated with PEI may be a potential gene carrier with high transfection efficacy for cancer gene therapy..

Reduction of transforming growth factor-β1 expression in leukemia and its possible role in leukemia development.

PubMed

Wu, Yong; Chen, Ping; Huang, Hui-Fang; Huang, Mei-Juan; Chen, Yuan-Zhong

2012-01-01

The expression of transforming growth factor-β1 (TGF-β1) in leukemic cells and sera from patients with leukemia and its possible role in leukemia development were studied. TGF-β1 levels in culture supernatants from leukemic cells were significantly lower than those from normal bone marrow mononuclear cells. Serum TGF-β1 levels in leukemic patients were significantly lower compared with healthy controls, but returned to normal in patients achieving complete remission, and decreased when patients relapsed. TGF-β1 mRNA expression levels were significantly higher in normal bone marrow mononuclear cells but lower in leukemic cells compared with normal CD34 + cells. After transfection of the TGF-β1 gene to HL-60 cells, cell apoptosis was detected. Moreover, by flow cytometry analysis, cells arrested in G1 phase were 62% for TGF-β1 transfected cells and 44% for controls. Transfection of exogenous TGF-β1 gene inhibited HL60 cells xenograft growth in nude mice, and prolonged survival of tumor-bearing mice compared with the controls. Decreased endogenous TGF-β1 expression in leukemia cells may be involved in leukemia development, Transfection of exogenous TGF-B1 gene to HL60 can inhibit the proliferation of the cells and induce cell apoptosis by down regulating bcl-2, hTERT (human telomerase reverse transcriptase) and c-myc expression.

Cellular uptake and ex vivo urothelial penetration by oligodeoxynucleotides for optimizing treatment of transitional cell carcinoma.

PubMed

Bolenz, Christian; Trojan, Lutz; Gabriel, Ute; Honeck, Patrick; Wendt-Nordahl, Gunnar; Schaaf, Axel; Alken, Peter; Michel, Maurice Stephan

2008-10-01

To evaluate cellular uptake and urothelial penetration of oligodeoxynucleotides (ODNs) in transitional cell carcinoma (TCC) cell lines and in a porcine ex vivo model, respectively. A panel of human TCC cell lines (RT 112, HT 1197 and UM-UC3) were exposed tofluorescein-labeled ODNs. Transfection rates were assessed byfluorescence microscopy and fluorescence-activated cell sorting (FACS). Intravesical treatment with ODNs was performed in a porcine ex vivo model. Urothelial penetration was evaluated using fluorescence microscopy of cryosections. Treatment with ODNs provided transfection rates of at least 96.8% of TCC cells, irrespective of use of a transfection agent. Effective urothelial penetration by ODNs was detected when compared with controls (p = 0.0325). The addition of a liposomal transfection agent significantly increased the penetration depth, allowing affection of deep urothelial cell layers (p = 0.0082). High transfection rates of ODNs can be achieved in TCC cells. Urothelial penetration of ODNs was observed down to the deepest cell layers when a transfection agent is added, suggesting a high potential for complementing the chemoresection effects on residual tumor areas during intravesical therapy of non-muscle-invasive TCC.

Transfection of cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat-shock-promoter-chloramphenicol-acetyltransferase construct.

PubMed

Stiles, B; Heilmann, J; Sparks, R B; Santoso, A; Leopold, R A

1992-01-01

Expression of heat shock proteins (hsp) in the BRL-AG-3C cell line from the cotton boll weevil was examined. It was determined that the maximal expression of endogenous hsp occurred at 41 degrees C. Various transfection methods were then compared using this cell line in conjunction with a transiently expressed bacterial gene marker (chloramphenicol acetyltransferase) which was under the control of the Drosophila hsp 70 gene promoter. The cationic lipid preparation Lipofectin was found to be very efficient at transfecting the boll weevil cells. Polylysine and 20-hydroxyecdysone-conjugated polylysine were moderately effective, whereas polybrene and electroporation, under the conditions reported herein, were ineffective at transfecting this cell line.

PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling

PubMed Central

2012-01-01

Background PCA3 is a non-coding RNA (ncRNA) that is highly expressed in prostate cancer (PCa) cells, but its functional role is unknown. To investigate its putative function in PCa biology, we used gene expression knockdown by small interference RNA, and also analyzed its involvement in androgen receptor (AR) signaling. Methods LNCaP and PC3 cells were used as in vitro models for these functional assays, and three different siRNA sequences were specifically designed to target PCA3 exon 4. Transfected cells were analyzed by real-time qRT-PCR and cell growth, viability, and apoptosis assays. Associations between PCA3 and the androgen-receptor (AR) signaling pathway were investigated by treating LNCaP cells with 100 nM dihydrotestosterone (DHT) and with its antagonist (flutamide), and analyzing the expression of some AR-modulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2 and PMEPA1). PCA3 expression levels were investigated in different cell compartments by using differential centrifugation and qRT-PCR. Results LNCaP siPCA3-transfected cells significantly inhibited cell growth and viability, and increased the proportion of cells in the sub G0/G1 phase of the cell cycle and the percentage of pyknotic nuclei, compared to those transfected with scramble siRNA (siSCr)-transfected cells. DHT-treated LNCaP cells induced a significant upregulation of PCA3 expression, which was reversed by flutamide. In siPCA3/LNCaP-transfected cells, the expression of AR target genes was downregulated compared to siSCr-transfected cells. The siPCA3 transfection also counteracted DHT stimulatory effects on the AR signaling cascade, significantly downregulating expression of the AR target gene. Analysis of PCA3 expression in different cell compartments provided evidence that the main functional roles of PCA3 occur in the nuclei and microsomal cell fractions. Conclusions Our findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in part through modulating AR signaling, which may raise new possibilities of using PCA3 knockdown as an additional therapeutic strategy for PCa control. PMID:23130941

Genetic Dissection of PTEN Signaling Mechanisms in Prostate Cancer

DTIC Science & Technology

2005-03-01

Renilla luciferase reporter to be used as a control for cell number and transfection efficiency. Luciferase assays were performed using the Dual...Luciferase Assay Kit from Promega. Reporter activities were normalized with the TK- Renilla luciferase control obtained from Promega. All assays were...Invitrogen). In addition to these components all transfections included 100 nanograms on the TK- Renilla luciferase reporter to be used as a control for cell

Natural Killer Cell Cytotoxicity Against SKOV3 after HLA-G Downregulation by shRNA.

PubMed

Nazari, Nazanin; Farjadian, Shirin

2016-09-01

HLA-G is a nonclassical HLA class I molecule which, when elevated in tumor cells, is one of the main factors involved in tumor evasion of immune responses including NK and T cells. To evaluate the effect of HLA-G downregulation on NK cell cytotoxicity in tumor cell lines. The expression level of HLA-G was measured by real-time PCR and flowcytometry after transfection of SKOV3 by shRNA.1, which targets specific sequences in exon 4, or shRNA.2, which targets both exons 4 and 6. NK-92MI cell cytotoxicity against transfected or untransfected target cell lines was measured with the lactate dehydrogenase (LDH) release assay. The Jeg-3 cell line was used as a positive control. Membrane-bound HLA-G expression levels decreased significantly in both cell lines after transfection with both shRNAs compared to their corresponding untransfected cells (p<0.05). Jeg-3 cells were better lysed than SKOV3 cells by NK cells during the first 48 h after transfection with both shRNAs. Compared to untransfected cells, shRNA.1-transfected SKOV3 cells were significantly more lysed by NK cells 24 h post-transfection (p=0.043). As a clinical approach, HLA-G downregulation with shRNA may be effective in cancer therapy by improving immune cell activation.

Reverse Transfection Using Gold Nanoparticles

NASA Astrophysics Data System (ADS)

Yamada, Shigeru; Fujita, Satoshi; Uchimura, Eiichiro; Miyake, Masato; Miyake, Jun

Reverse transfection from a solid surface has the potential to deliver genes into various types of cell and tissue more effectively than conventional methods of transfection. We present a method for reverse transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagentcomplex on a glass surface, which could then be used for the regulation of the particle size of the complex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet-PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared with the control without GC. In this manner, we constructed a method for reverse transfection using GC to deliver genes into the cells effectively.

Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

PubMed

Cervera, Laura; Kamen, Amine A

2018-01-01

Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1 × 10 6 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts.

PubMed

Li, Li-Hua; Guo, Zi-Jian; Yan, Ling-Ling; Yang, Ji-Cheng; Xie, Yu-Feng; Sheng, Wei-Hua; Huang, Zhao-Hui; Wang, Xue-Hao

2007-12-21

To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7,721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed. VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7,721 cells and xenografted tumor. Compared to the control group, the transfected cells grew slower in cell cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

Re-induction of cell differentiation and (131)I uptake in dedifferentiated FTC-133 cell line by TSHR gene transfection.

PubMed

Feng, Fang; Wang, Hui; Hou, Shasha; Fu, Hongliang

2012-11-01

Radioiodine therapy is commonly used to treat differentiated thyroid cancer (DTC), but a major challenge is dedifferentiation of DTC with the loss of radioiodine uptake. TSHR is a key molecule regulating thyrocyte proliferation and function. This study aimed to test the therapeutic potential of TSHR in dedifferentiated DTC by gene transfection in order to restore cell differentiation and radioiodine uptake. Dedifferentiated FTC-133 (dFTC-133) cells were obtained by monoclonal culture of FTC-133 cell line after (131)I radiation. Recombinant plasmid pcDNA3.1-hTSHR was transfected into dFTC-133 cells by using Lipofectamine 2000 reagent. Immunofluorescence analysis was carried out to confirm TSHR expression and its location. Radioiodine uptake assay was thereafter investigated. mRNAs and proteins of TSHR and other thyroid differentiated markers were detected by real-time PCR and western blot respectively. Among the thyroid specific genes in dFTC-133 cells with stable low radioiodine uptake, TSHR was down-regulated most significantly compared with FTC-133. Then, after TSHR gene transfection, augmented expression of TSHR was observed in dFTC-133 cell surface and cytoplasm by immunofluorescence analysis. It was found that (125)I uptake was 2.9 times higher (t=28.63, P<.01) in cells with TSHR transfection than control. The mRNAs of TSHR, NIS, TPO and Tg were also significantly increased by 1.7 times (t=13.8, P<.05), 4 times (t=28.52, P<.05), 1.5 times (t=14.43, P<.05) and 2.2 times (t=19.83, P<.05) respectively compared with control group. Decreased TSHR expression correlated with FTC-133 ongoing dedifferentiation. TSHR transfection contributed to the re-differentiation of dedifferentiated thyroid follicular carcinoma cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibition of subcutaneously implanted human pituitary tumor cells in nude mice by LRIG1.

PubMed

Wang, X; He, X J; Xu, H Q; Chen, Z W; Fan, H H

2016-05-06

The aim of this study was to explore the inhibition of subcutaneously implanted human pituitary tumor cells in nude mice by LRIG1 and its mechanism. For this study, athymic nude mice were injected with either normal pituitary tumor RC-4B/C cells or LRIG1-transfected RC-4B/C cells. We then calculated the volume inhibition rate of the tumors, as well as the apoptosis index of tumor cells and the expression of Ras, Raf, AKt, and ERK mRNA in tumor cells. Tumor cell morphological and structural changes were also observed under electron microscope. Our data showed that subcutaneous tumor growth was slowed or even halted in LRIG1-transfected tumors. The tumor volumes were significantly different between the two groups of mice (χ2 = 2.14, P < 0.05). The tumor apoptosis index was found to be 8.72% in the control group and 39.7% in LRIG1-transfected mice (χ2 = 7.59, P < 0.05). The levels of Ras, Raf, and AKt mRNA in LRIG1-transfected RC-4B/C cells were significantly reduced after transfection (P < 0.01). Transfected subcutaneous tumor cells appeared to be in early or late apoptosis under an electron microscope, while only a few subcutaneous tumor cells appeared to be undergoing apoptosis in the control group. In conclusion, the LRIG1 gene is able to inhibit proliferation and promote apoptosis in subcutaneously implanted human pituitary tumors in nude mice. The mechanism of LRIG1 may involve the inhibition of the PI3K/ Akt and Ras/Raf/ERK signal transduction pathways.

Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

NASA Astrophysics Data System (ADS)

Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

2007-12-01

Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray spots carrying different DNA vectors was also investigated. By application of a voltage of 286 V/cm for 10 ms, transfection efficiency was doubled compared to using only transfection reagent, whilst maintaining a cell viability of 60-70% of the positive control.

Effects of gene silencing of CypB on gastric cancer cells.

PubMed

Guo, Feng; Zhang, Ying; Zhao, Chun-Na; Li, Lin; Guo, Yan-Jun

2015-04-01

To determine the effect of gene silencing of cyclophilin B (CypB) on growth and proliferation of gastric cancer cells. CypB siRNA lentivirus (LV-CypB-si) and control lentivirus (LV-si-con) were produced. CypB expression in gastric cancer cell lines was detected by Western blot. BGC823 and SGC7901 cells were chosen to be infected with LV-si-con and LV-CypB-si, and stable transfectants were isolated. The cell groups transfected with LV-CypB-siRNA, LV-siRNA-con and transfected no carrier were served as the experimental group, the implicit control group and the blank control group respectively. MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro. Cell cycle was analyzed with flow cytometry. The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot. Gene silencing of CypB can inhibit gastric cancer cell growth, proliferation, cell cycle progress and tumorigenesis. CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES. These two cell lines were infected with LV-si-con and LV-CypB-si respectively. MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si (P<0.05). Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G1 (P<0.05). These findings indicated that CypB could promote the G1-S transition of gastric cancer cell. In addition, the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group. Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis. These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Development of an electro-responsive platform for the controlled transfection of mammalian cells

NASA Astrophysics Data System (ADS)

Hook, Andrew L.; Thissen, Helmut W.; Hayes, Jason P.; Voelcker, Nicolas H.

2005-02-01

The recent development of living microarrays as novel tools for the analysis of gene expression in an in-situ environment promises to unravel gene function within living organisms. In order to significantly enhance microarray performance, we are working towards electro-responsive DNA transfection chips. This study focuses on the control of DNA adsorption and desorption by appropriate surface modification of highly doped p++ silicon. Silicon was modified by plasma polymerisation of allylamine (ALAPP), a non-toxic surface that sustains cell growth. Subsequent high surface density grafting of poly(ethylene oxide) formed a layer resistant to biomolecule adsorption and cell attachment. Spatially controlled excimer laser ablation of the surface produced micron resolution patterns of re-exposed plasma polymer whilst the rest of the surface remained non-fouling. We observed electro-stimulated preferential adsorption of DNA to the ALAPP surface and subsequent desorption by the application of a negative bias. Cell culture experiments with HEK 293 cells demonstrated efficient and controlled transfection of cells using the expression of green fluorescent protein as a reporter. Thus, these chemically patterned surfaces are promising platforms for use as living microarrays.

Gene therapy of uterine leiomyoma: adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir treatment inhibits growth of human and rat leiomyoma cells in vitro and in a nude mouse model.

PubMed

Salama, S A; Kamel, M; Christman, G; Wang, H Q; Fouad, H M; Al-Hendy, A

2007-01-01

Uterine leiomyomas (LM) affect a high percentage of reproductive-age women. They develop as discrete, well-defined tumors that are easily accessible with imaging techniques--making this disease ideal for localized gene therapy approaches. In this study, we determined the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene transfer in combination with ganciclovir (Ad-TK/GCV) as a potential therapy for LM. Rat ELT-3 LM cells and human LM cells were transfected with different multiplicity of infections (10-100 plaque forming units [PFU]/cell) of Ad-TK and treated with GCV (5, 10, or 20 microg/ml) for 5 days. To test the bystander effect, Ad-TK-transfected ELT-3 cells (100 PFU/cell) or LM cells (10 PFU/cell) were cocultured with corresponding nontransfected cells at increasing percentages and treated with GCV followed by cell counting. In ELT-3 cells transfected with Ad-TK/GCV (10, 20, 50, or 100 PFU/cell), the cell count was reduced by 24, 42, 77, and 87%, respectively, compared with the control cells (transfected with Ad-Lac Z/GCV). Similarly, in LM cells transfected with Ad-TK/GCV (10, 50, or 100 PFU/cell), the cell count was reduced by 31, 62, and 82%, respectively, compared with the control. A strong bystander effect was noted in both ELT-3 and LM cells with significant killing (p = 0.001) at a ratio of infected:uninfected cells of only 1:99 and maximal killing at 1:4. This study demonstrates the potential efficacy of the Ad-TK/GCV gene therapy approach as a viable nonsurgical alternative treatment for uterine LM.

Transcriptional regulation of human retinoic acid receptor-alpha (RAR-{alpha}) by Wilms` tumour gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodyer, P.R.; Torban, E.; Dehbi, M.

1994-09-01

The Wilms` tumor gene encodes a 47-49 kDa transcription factor expressed in kidney, gonads and mesothelium during embryogenesis. Inherited mutations of WT1 lead to aberrant urogenital development and Wilms` tumor, but the role of WT1 in development is not fully understood. Since the human RAR-{alpha} gene contains a potential WT1 binding site at its 5{prime} end, we studied the effect of WT1 co-transfection on expression of an RAR-{alpha} promoter/CAT reporter construct in COS cells. COS cells were plated at 5X10{sup 5} cells/dish in DMEM with 10% FBS and transfected by the Ca/PO4 method with an expression plasmid containing the full-lengthmore » WT1 (-/-) cDNA under the control of the CMV promoter, plasmid containing the RAR-{alpha} promoter (-519 to +36)/CAT reporter and TK/growth hormone plasmid to control for efficiency of transfection. CAT/GH activity at 48 hours was inhibited by co-transfection with increasing amounts of WT1 (-/-); maximum inhibition = 5% of control. WT1 co-transfection did not affect expression of TKGH, nor of a CMV-CAT vector. Expression of WT1 protein in tranfected COS cells was demonstrated by Western blotting. Minimal inhibiton of RAR-{alpha}/CAT activity was seen when cells were co-transfected with vectors containing WT1 deletion mutants, alternate WT1 splicing variants, or WT1 (-/-) cDNA bearing a mutation identified in a patient with Drash syndrome. Gel shift assays indicated binding of WT1 to RAR-{alpha} cDNA but not to an RAR-{alpha} deletion mutant lacking the GCGGGGGGCG site. These observations suggest that WT1 may function to regulate RAR-{alpha} expression during normal development.« less

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

PubMed

Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

2017-02-01

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. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Osteoprotegerin Promotes Cementoblastic Activity of Murine Cementoblast Cell Line in vitro.

PubMed

Zhang, Ying Ying; Zhao, Hua Xiang; Chen, Zhi Bin; Lin, Jiu Xiang; Liu, Yan

2016-06-01

To investigate the effect of osteoprotegerin (OPG) on the cementoblastic activity of a clonal population of immortalised murine cementoblasts (OCCM-30) in vitro. OCCM-30 cells were transiently transfected with the mouse OPG using the Avalanche transfection reagent. The ectopic expression of OPG was confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. The cell counting Kit-8 assay was used to investigate the effect of OPG on cell proliferation. The expression levels of cementoblastic-related mRNA and protein in the transfected OCCM-30 cells were detected using real-time PCR, Western blotting and immunohistochemical staining. Satisfactory transfection efficiency was observed 48 h after transfection. The results of the cell proliferation assay indicated that the expansion rate of the OPG transfection group was greater than that of the control group at both 72 h and 96 h. The mRNA levels of osterix (Osx), protein kinase B (Akt1), cementum attachment protein (CAP) and osteopontin (Opn) were significantly upregulated (P < 0.05) in the OPG group. Protein levels of OPN, bone sialoprotein II (BSP II), osteocalcin (OC) and CAP, which are responsible for osteogenetic and cementoblastic activity, were significantly increased in the OPG-overexpressing group. Overexpression of OPG in OCCM-30 cells promotes cementoblastic activity.

Enhancing the Breadth of Efficacy of Therapeutic Vaccines for Breast Cancer

DTIC Science & Technology

2015-10-01

from recombinant baculovirus- infected insect cells . *Currently expressing antigens in C1R cells since these cells can also be used as antigen...co-transfect SF9 insect cells . Co-transfection of SF9 cells is initially assesed by survival of the cells compared to un-infected controls. The...Clones with higher TCR production are amplified again and used to infect Hi5 insect cells for protein production. 17 Figure 10. ELISA used to

MicroRNA-214 protects against hypoxia/reoxygenation induced cell damage and myocardial ischemia/reperfusion injury via suppression of PTEN and Bim1 expression.

PubMed

Wang, Xiaohui; Ha, Tuanzhu; Hu, Yuanping; Lu, Chen; Liu, Li; Zhang, Xia; Kao, Race; Kalbfleisch, John; Williams, David; Li, Chuanfu

2016-12-27

Myocardial apoptosis plays an important role in myocardial ischemia/reperfusion (I/R) injury. Activation of PI3K/Akt signaling protects the myocardium from I/R injury. This study investigated the role of miR-214 in hypoxia/reoxygenation (H/R)-induced cell damage in vitro and myocardial I/R injury in vivo. H9C2 cardiomyoblasts were transfected with lentivirus expressing miR-214 (LmiR-214) or lentivirus expressing scrambled miR-control (LmiR-control) respectively, to establish cell lines of LmiR-214 and LmiR-control. The cells were subjected to hypoxia for 4 h followed by reoxygenation for 24 h. Transfection of LmiR-214 suppresses PTEN expression, significantly increases the levels of Akt phosphorylation, markedly attenuates LDH release, and enhances the viability of the cells subjected to H/R. In vivo transfection of mouse hearts with LmiR-214 significantly attenuates I/R induced cardiac dysfunction and reduces I/R-induced myocardial infarct size. LmiR-214 transfection significantly attenuates I/R-induced myocardial apoptosis and caspase-3/7 and caspase-8 activity. Increased expression of miR-214 by transfection of LmiR-214 suppresses PTEN expression, increases the levels of phosphorylated Akt, represses Bim1 expression and induces Bad phosphorylation in the myocardium. In addition, in vitro data shows transfection of miR-214 mimics to H9C2 cells suppresses the expression and translocation of Bim1 from cytosol to mitochondria and induces Bad phosphorylation. Our in vitro and in vivo data suggests that miR-214 protects cells from H/R induced damage and attenuates I/R induced myocardial injury. The mechanisms involve activation of PI3K/Akt signaling by targeting PTEN expression, induction of Bad phosphorylation, and suppression of Bim1 expression, resulting in decreases in I/R-induced myocardial apoptosis.

Small interfering RNA-mediated suppression of serum response factor, E2-promotor binding factor and survivin in non-small cell lung cancer cell lines by non-viral transfection.

PubMed

Walker, Tobias; Nolte, Andrea; Steger, Volker; Makowiecki, Christina; Mustafi, Migdat; Friedel, Godehard; Schlensak, Christian; Wendel, Hans-Peter

2013-03-01

Serum response factor (SRF), E2F1 and survivin are well-known factors involved in a multitude of cancer-related regulation processes. However, to date, no suitable means has been found to apply their potential in the therapy of non-small cell lung cancer (NSCLC). This study deals with questions of small interfering ribonucleic acid (siRNA) transfection efficiency by a non-viral transfection of NSCLC cell-lines and the power of siRNA to transiently influence cell division by specific silencing. Different NSCLC cell lines were cultured under standard conditions and transfected, with specific siRNA targeting SRF, E2F1 and survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as controls. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for messenger RNA (mRNA) expression levels. Additionally, transfection efficiency was evaluated by flow cytometry. The analysis of cell proliferation was determined with a CASY cell counter 3 days after transfection with SRF or SCR-siRNA. Transfection of the NSCLC cell lines with specific siRNAs against SRF, E2F1 and survivin resulted in a very considerable reduction of the intracellular mRNA concentration. CASY confirmation of cell viability demonstrated an excellent survival of the cell lines treated with non-specific siRNA, in contrast to with application of specific siRNA. This study reports a reliable transfectability of NSCLC-cell lines by siRNA, initially in a non-viral manner, and a reproducible knockdown of the focussed targets, consequently leading to the death of the tumour cells. This constitutes a strong candidate for a new assessment strategy in the therapy of non-small cell lung cancer.

[Effects of parabolic flight on redox status in SH-SY5Y cells].

PubMed

Bi, Lei; Qu, Li-Na; Huang, Zeng-Ming; Wang, Chun-Yan; Li, Qi; Tan, Ying-Jun; Li, Ying-Hui

2009-10-25

Space flight is known to produce a number of neurological disturbances. The etiology is unknown, but it may involve increased oxidative stress. A line of experimental evidence indicates that space flight may disrupt antioxidant defense system and result in increased oxidative stress. In vitro studies found that abundant of NO was produced in rat pheochromocytoma (PC12) cells, SHSY5Y neuroblastoma cells, and protein nitration was increased in PC12 cells within a simulated microgravity rotating wall bioreactor high aspect ratio vessel system or clinostat system. In the present study, we observed the change of redox status in SH-SY5Y cells after parabolic flight, and studied the effects of key redox molecule, thioredoxin (TRX), during the altered gravity. SH-SY5Y cells were divided into four groups: control cells, control cells transfected with TRX, flight cells and flight cells transfected with TRX. The expression levels of 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), TRX and thioredoxin reductase (TRXR) were observed by immunocytochemical method. It was shown that after parabolic flight, the staining of 3-NT and TRX were enhanced, while the expression level of TRXR was down-regulated compared with control. As for flight cells transfected with TRX, the staining of 3-NT and iNOS were weakened compared with flight cells. These results obtained suggest that altered gravity may increase protein nitration, down-regulate TRXR and elicit oxidative stress in SH-SY5Y cells, while TRX transfection could partly protect cells against oxidative stress induced by parabolic flight.

The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver.

PubMed

Meijer, Joost; Zeelenberg, Ingrid S; Sipos, Bence; Roos, Ed

2006-10-01

The chemokine receptor CXCR5 is expressed by B cells and certain T cells and controls their migration into and within lymph nodes. Its ligand BCA-1/CXCL13 is present in lymph nodes and spleen and also in the liver. Surprisingly, we detected CXCR5 in several mouse and human carcinoma cell lines. CXCR5 was particularly prominent in pancreatic carcinoma cell lines and was also detected by immunohistochemistry in 7 of 18 human pancreatic carcinoma tissues. Expression in CT26 colon carcinoma was low in vitro, up-regulated in vivo, and rapidly lost when cells were explanted in vitro. CXCL13 strongly promoted proliferation of CXCR5-transfected CT26 cells in vitro. In the liver, after intrasplenic injection, these CXCR5 transfectants initially grew faster than controls, but the growth rate of control tumors accelerated later to become similar to the transfectants, likely due to the up-regulation of CXCR5. Inhibition of CXCR5 function, by trapping CXCR5 in the endoplasmic reticulum using a CXCL13-KDEL "intrakine," had no effect on initial growth of liver foci but later caused a prolonged growth arrest. In contrast, s.c. and lung tumors of CXCR5- and intrakine-transfected cells grew at similar rates as controls. We conclude that expression of CXCR5 on tumor cells promotes the growth of tumor cells in the liver and, at least for CT26 cells, seems to be required for outgrowth to large liver tumors. Given the limited expression on normal cells, CXCR5 may constitute an attractive target for therapy, particularly for pancreatic carcinoma.

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

PubMed Central

Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

2016-01-01

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity. PMID:26843283

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

NASA Astrophysics Data System (ADS)

Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

2016-02-01

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro.

PubMed

Ma, Y F; Wu, Z H; Gao, M; Loor, J J

2018-06-01

The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H 2 O 2 -induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H 2 O 2 (600 μM) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H 2 O 2 , respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H 2 O 2 compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H 2 O 2 compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H 2 O 2 compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6-XL5-NFE2L2 and without H 2 O 2 . However, compared with the control, production of ROS and MDA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H 2 O 2 . Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H 2 O 2 had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H 2 O 2 had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H 2 O 2 had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H 2 O 2 compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

WNT/β-catenin pathway is modulated in asthma patients and LPS-stimulated RAW264.7 macrophage cell line.

PubMed

Lee, Haeyong; Bae, Sungmin; Choi, Byoung Whui; Yoon, Yoosik

2012-02-01

In the present study, we investigated the possibility that the WNT/β-catenin pathway plays a role in inflammatory responses both in an human inflammatory condition and in an in vitro inflammation model. First, we analyzed gene expression patterns of the peripheral blood cells from asthma patients compared with those from normal subjects using microarray analyses. We found that intracellular signaling molecules of the WNT/β-catenin pathway were significantly changed in asthma patients compared with the levels in the controls. Next, we determined whether major components of the WNT/β-catenin pathway were involved in the lipopolysaccharide (LPS)-induced inflammatory response of the RAW264.7 macrophage cell line. Among the members of WNT/β-catenin pathway, the protein levels of low-density lipoprotein receptor-related protein (LRP) 6, dishevelled (DVL) 2, and AXIN1, which were measured using western blotting, did not significantly change in the presence of LPS. In contrast, the LPS induced a rapid phosphorylation of glycogen synthase kinase (GSK) 3β and accumulation of β-catenin protein. It was found that β-catenin plays a significant role in the LPS-induced inflammatory response through the performance of small interfering RNA (siRNA) transfection experiments. The mRNA level of IL-6 was significantly elevated in β-catenin siRNA-transfected cells compared with that in control siRNA-transfected cells after LPS treatment. Furthermore, nuclear factor-κB (NF-κB) activity was also significantly increased in β-catenin siRNA-transfected cells compared with the level seen in control siRNA-transfected cells. Taken together, these results suggest that β-catenin plays a role as a negative regulator, preventing the overproduction of inflammatory cytokines such as IL-6 in LPS-induced inflammatory responses.

IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma.

PubMed

Ueno, Koji; Hirata, Hiroshi; Majid, Shahana; Tabatabai, Z Laura; Hinoda, Yuji; Dahiya, Rajvir

2011-11-15

The Wnt/β-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ β-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and β-catenin expression was increased. Also the β-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis. Copyright © 2011 UICC.

Effects of siRNA-mediated suppression of HPV-11 L1 expression on the proliferation and apoptosis of vaginal epithelial cells

PubMed Central

Zeng, Juan; Yang, Shumei; Wang, Xiaorui; Gao, Yan; Zhang, Mei

2017-01-01

The aim of the present study was to investigate the effects of human papillomavirus (HPV) infection on the gynecological disease of vaginitis and to demonstrate how the small interfering RNA (siRNA) method may be used for HPV prevention in the clinic. Human vaginal epithelial cells were transfected with HPV-11 L1 expression vector and siRNA-HPV-11 L1 vectors and a control group was transfected with scrambled siRNA. Cell proliferation in each group was analyzed using the MTT assay and the expression of apoptosis-associated proteins was measured by western blot analysis. Compared with the control group, HPV-11 L1 mRNA and protein levels were significantly increased following transfection with the HPV-11 L1 expression vector in cells (P<0.05), but this result was significantly reversed by silencing of HPV-11 L1 (P<0.05). In addition, cell proliferation in the HPV-11 group was lower than that in the control group; however, cell proliferation was significantly increased in cells transfected with silenced L1 compared with that in the control group (P<0.05). Furthermore, silencing of HPV-11 L1 significantly decreased caspase-3 and caspase-9 expressions in cells, whereas the expression was increased in the HPV-11 L1 group (P<0.05). The present study suggested that siRNA-mediated silencing of HPV-11 L1 may have potential therapeutic applications for treating gynecological diseases associated with HPV-11 infection. PMID:28413509

Immunobiologic effects of cytokine gene transfer of the B16-BL6 melanoma.

PubMed

Strome, S E; Krauss, J C; Cameron, M J; Forslund, K; Shu, S; Chang, A E

1993-12-01

The genetic modification of tumors offers an approach to modulate the host immune response to relatively weak native tumor antigens. We examined the immunobiologic effects of various cytokine genes transferred into the poorly immunogenic B16-BL6 murine melanoma. Retroviral expression vectors containing cDNAs for interleukin 2, interleukin 4, interferon gamma, or a neomycin-resistant control were electroporated into a B16-BL6 tumor clone. Selected transfected clones were examined for in vitro cytokine secretion and in vivo tumorigenicity. When cells from individual clones were injected intradermally into syngeneic mice, the interleukin 4-secreting clone grew significantly slower than did the neomycin-resistant transfected control, while the growth of the interleukin 2- and interferon gamma-expressing clones was not affected. Despite minimal cytokine secretion by interferon gamma-transfected cells, these cells expressed upregulated major histocompatibility class I antigen and were more susceptible to lysis by allosensitized cytotoxic T lymphocytes compared with parental or neomycin-resistant transfected tumor targets. We observed diverse immunobiologic effects associated with cytokine gene transfer into the B16-BL6 melanoma. Interleukin 4 transfection of tumor resulted in decreased in vivo tumorigenicity that may be related to a host immune response. Further studies to evaluate the host T-cell response to these gene-modified tumors are being investigated.

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

PubMed

Mie, Masayasu; Endoh, Tamaki; Yanagida, Yasuko; Kobatake, Eiry; Aizawa, Masuo

2003-02-13

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

[Construction of injectable tissue engineered adipose tissue with fibrin glue scaffold and human adipose-derived stem cells transfected by lentivirus vector expressing hepatocyte growth factor].

PubMed

Zhu, Yuanzheng; Yi, Yangyan; Yang, Shuifa; Zhang, Jing; Wu, Shu; Wang, Zhaohui

2017-09-01

To discuss the possibility of constructing injectable tissue engineered adipose tissue, and to provide a new approach for repairing soft tissue defects. Human adipose-derived stem cells (hADSCs) were extracted from the lipid part of human liposuction aspirate by enzymatic digestion and identified by morphological observation, flow cytometry, and adipogenic induction. The hADSCs underwent transfection by lentivirus vector expressing hepatocyte growth factor and green fluorescent protein (HGF-GFP-LVs) of different multiplicity of infection (MOI, 10, 30, 50, and 100), the transfection efficiency was calculated to determine the optimum MOI. The hADSCs transfected by HGF-GFP-LVs of optimal MOI and being adipogenic inducted were combined with injectable fibrin glue scaffold, and were injected subcutaneously into the right side of the low back of 10 T-cell deficiency BALB/c female nude mice (transfected group); non-HGF-GFP-LVs transfected hADSCs (being adipogenic inducted) combined with injectable fibrin glue scaffold were injected subcutaneously into the left side of the low back (untransfected group); and injectable fibrin glue scaffold were injected subcutaneously into the middle part of the neck (blank control group); 0.4 mL at each point. Twelve weeks later the mice were killed and the implants were taken out. Gross observation, wet weight measurement, HE staining, GFP fluorescence labeling, and immunofluorescence staining were performed to assess the in vivo adipogenic ability of the seed cells and the neovascularization of the grafts. The cultured cells were identified as hADSCs. Poor transfection efficiency was observed in MOI of 10 and 30, the transfection efficiency of MOI of 50 and 100 was more than 80%, so the optimum MOI was 50. Adipose tissue-like new-born tissues were found in the injection sites of the transfected and untransfected groups after 12 weeks of injection, and no new-born tissues was found in the blank control group. The wet-weight of new-born tissue in the transfected group [(32.30±4.06) mg] was significantly heavier than that of the untransfected group [(25.27±3.94) mg] ( t =3.929, P =0.001). The mature adipose cells in the transfected group [(126.93±5.36) cells/field] were significantly more than that in the untransfected group [(71.36±4.52) cells/field] ( t =30.700, P =0.000). Under fluorescence microscopy, some of the single cell adipocytes showed a network of green fluorescence, indicating the presence of GFP labeled exogenous hADSCs in the tissue. The vascular density of new-born tissue of the transfected group [(16.37±2.76)/field] was significantly higher than that of the untransfected group [(9.13±1.68)/field] ( t =8.678, P =0.000). The hADSCs extracted from the lipid part after liposuction can be used as seed cells. After HGF-GFP-LVs transfection and adipose induction, the hADSCs combined with injectable fibrin glue scaffold can construct mature adipose tissue in vivo , which may stimulate angiogenesis, and improve retention rate of new-born tissue.

The feasibility of using magnetic nanoparticles modified as gene vector.

PubMed

Chen, D; Tang, Q; Xue, W; Wang, X

2010-06-01

To evaluate the feasibility of using magnetic nanoparticles (MNPs) as gene vector and the effect of magnetic field on efficiency of transfection. Magnetic nanoparticles were prepared by controlling some chemical reaction parameters through a partially reduction precipitation method with ferric chloride aqueous solution as precursor material. The surface of particles was modified by polyethyleneimine (PEI) agents. The appearance, the size distribution, structure and phase constitute of MNPs were characterized by Transmission electron microscope (TEM), X-ray diffraction (XRD); the potential of absorbing DNA of MNPs was analysed by electrophoresis. Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using MNPs-PLL as vector. The effect of magnetic field on the efficiency of transfection was determined using Nd-Fe-B permanent magnet. Foreign gene could be delivered to various cell lines by MNPs-PLL and expressed with high efficiency but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5-10 fold. MNPs- PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Proteome alteration induced by hTERT transfection of human fibroblast cells.

PubMed

Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions.

Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs.

PubMed

Tu, Tran Cam; Yamashita, Toshiharu; Kato, Toshiki; Nagano, Masumi; Trinh, Nhu Thuy; Hamada, Hiromi; Sato, Fujio; Ohneda, Kinuko; Matsuo-Takasaki, Mami; Ohneda, Osamu

2016-08-12

Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

PubMed

Rajendra, Yashas; Balasubramanian, Sowmya; Kiseljak, Divor; Baldi, Lucia; Wurm, Florian M; Hacker, David L

2015-01-01

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection. © 2015 American Institute of Chemical Engineers.

[The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes].

PubMed

Huang, Ji-yan; Liu, Wei; Zhou, Zeng-tong; Zhou, Hai-wen

2014-06-01

Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA

PubMed Central

Sridharan, Arati; Patel, Chetan; Muthuswamy, Jit

2013-01-01

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after −1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after −1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at −1 V compared with the same at ±2–3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons. PMID:23531602

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA.

PubMed

Sridharan, Arati; Patel, Chetan; Muthuswamy, Jit

2013-03-26

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after -1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after -1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at -1 V compared with the same at ±2-3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons.Molecular Therapy-Nucleic Acids (2013) 2, e82; doi:10.1038/mtna.2013.10; published online 26 March 2013.

Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells.

PubMed

Khanizadeh, Sayyad; Ravanshad, Mehrdad; Hosseini, SeyedYounes; Davoodian, Parivash; Nejati Zadeh, Azim; Sarvari, Jamal

2015-01-01

In this study, to clarify the SMAD4 blocking impact on fibrosis process, we investigated its down-regulation by shRNA on activated human LX-2 cell, in vitro. Liver fibrosis is a critical consequence of chronic damage to the liver that can progress toward advanced diseases, liver cirrhosis and hepatocellular carcinoma (HCC). Different SMAD proteins play as major mediators in the fibrogenesis activity of hepatic stellate cells through TGF-β pathways, but the extent of SMAD4 as a co-SMAD protein remained less clear. vector expressing verified shRNA targeting human SMAD4 gene was transfected into LX-2 cells. The GFP expressing plasmid was transfected in the same manner as a control group while leptin treated cells were employed as positive controls. Subsequently, total RNA was extracted and real-time PCR was performed to measure the mRNA levels of SMAD4, COL-1A1, α-SMA, TGF-β and TIMP-1. Furthermore, trypan blue exclusion was performed to test the effect of plasmid transfection and SMAD4 shutting-down on cellular viability. The results indicated that the expression of SMAD4was down-regulated following shRNA transfection intoLX-2 cells (P<0.001). The gene expression analysis of fibrotic genes in LX-2 cells showed that SMAD4 blocking by shRNA significantly reduced the expression level of fibrotic genes when compared to control plasmids (P<0.001). Vector expressing SMAD4-shRNA induced no significant cytotoxic or proliferative effects on LX-2 cells as determined by viability assay (P<0.05). The results of this study suggested that knockdown of SMAD4 expression in stellate cell can control the progression of fibrogenesis through TGF-β pathway blocking.

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury.

PubMed

Munemasa, Yasunari; Chang, Chang-Sheng; Kwong, Jacky M K; Kyung, Haksu; Kitaoka, Yasushi; Caprioli, Joseph; Piri, Natik

2012-01-01

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).

The Neuronal EGF-Related Gene Nell2 Interacts with Macf1 and Supports Survival of Retinal Ganglion Cells after Optic Nerve Injury

PubMed Central

Munemasa, Yasunari; Chang, Chang-Sheng; Kwong, Jacky M. K.; Kyung, Haksu; Kitaoka, Yasushi; Caprioli, Joseph; Piri, Natik

2012-01-01

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01). PMID:22496866

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer.

PubMed

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-10-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer

PubMed Central

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-01-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer. PMID:28943918

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

2008-11-15

Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

Localized transfection on arrays of magnetic beads coated with PCR products.

PubMed

Isalan, Mark; Santori, Maria Isabel; Gonzalez, Cayetano; Serrano, Luis

2005-02-01

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Spatial Control of Cell Transfection Using Soluble or Solid-Phase Redox Agents and a Redox-Active Ferrocenyl Lipid

PubMed Central

Aytar, Burcu S.; Muller, John P. E.; Kondo, Yukishige; Abbott, Nicholas L.; Lynn, David M.

2013-01-01

We report principles for active, user-defined control over the locations and timing with which DNA is expressed in cells. Our approach exploits unique properties of a ferrocenyl cationic lipid that is inactive when oxidized, but active when chemically reduced. We show that methods that exert spatial control over the administration of reducing agents can lead to local activation of lipoplexes and spatial control over gene expression. The versatility of this approach is demonstrated using both soluble and solid-phase reducing agents. These methods provide control over cell transfection, including methods for remote activation and the patterning of expression using solid-phase redox agents, that are difficult to achieve using conventional lipoplexes. PMID:23965341

Spatial control of cell transfection using soluble or solid-phase redox agents and a redox-active ferrocenyl lipid.

PubMed

Aytar, Burcu S; Muller, John P E; Kondo, Yukishige; Abbott, Nicholas L; Lynn, David M

2013-09-11

We report principles for active, user-defined control over the locations and timing with which DNA is expressed in cells. Our approach exploits unique properties of a ferrocenyl cationic lipid that is inactive when oxidized, but active when chemically reduced. We show that methods that exert spatial control over the administration of reducing agents can lead to local activation of lipoplexes and spatial control over gene expression. The versatility of this approach is demonstrated using both soluble and solid-phase reducing agents. These methods provide control over cell transfection, including methods for remote activation and the patterning of expression using solid-phase redox agents, that are difficult to achieve using conventional lipoplexes.

Cell cycle distribution, cellular viability and mRNA expression of hGCase-gene-transfected cells in dairy goat.

PubMed

Zhang, Yan-Li; Wan, Yong-Jie; Wang, Zi-Yu; Qi, Wei-Wei; Zhou, Zheng-Rong; Huang, Rong; Wang, Feng

2010-05-07

Nuclear transfer using transgenic donor cells is an efficient way of generating transgenic goats, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. We have measured the efficiency of transfection with a plasmid containing hGCase (human lysosomal acid beta-glucosidase) gene into goat FFC (fetal-derived fibroblast cells), MEC (mammary epithelial cells) and AEFC (adult ear skin-derived fibroblast cells), and the characteristics of cell cycle, apoptosis and chromosome abnormalities after transfection. The expression of genes involved in imprinting [IGF2 (insulin-like growth factor 2), IGF2R (IGF2 receptor)], apoptosis (Bax), stress (heat-shock protein, Hsp70.1), cellular connections [Cx43 (connexin 43)] and DNA methylation [DNMT1 (DNA methyltransferase 1)] in transgenic fetal cells has been investigated. The hGCase transgene was successfully detected in the transfected cell lines, and chromosomal stability remained similar in FFC and transgenic FFC (70.9 compared with 66.8%), whereas a smaller percentage (P<0.05) of cells at G(0)/G(1) in the transgenic FFC, MEC and AEFC (T-FFC, T-MEC and T-AEFC), and higher percentage (P<0.05) of apoptotic cells in T-FFC than the non-transfected controls were detected by flow cytometric analysis. Among the genes tested, the relative expressions of IGF2, IGF2R and transcripts of Cx43 were significantly higher (P<0.05) in T-FFC compared with non-transfected FFC. These novel findings on gene expression in transgenic fetal cells may have certain implications in the biopharming industry and in our understanding the low efficiency of transgenic cloning.

[EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS].

PubMed

Fu, Weili; Chen, Gang; Tang, Xin; Li, Qi; Ll, Jian

2015-04-01

To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000). Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.

Molecular Determinants of Antiestrogen and Drug Sensitivity in Breast Carcinoma Cells

DTIC Science & Technology

1997-08-01

W.S. human HT1080 fibrosarcoma cells (a gift of Pear, personal communication). G.R. Stark), susceptible to ecotropic retrovirus In several...transfection with equal amounts of plasmid pOPRSVIluc (generated by replacing CAT of pORRSVICAT with luc) which expresses luc from R02 promoter, and control...retroviral transduction into (Fig. 1). During transient co-transfection of NIH 3T3 human HT1080 fibrosarcoma or mouse NIH 3T3 cell cells with a laac expressing

Transfection using DEAE-dextran.

PubMed

Gulick, T

2001-05-01

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

Transfection using DEAE-dextran.

PubMed

Gulick, Tod

2003-08-01

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

Transfection using DEAE-dextran.

PubMed

Gulick, T

2001-05-01

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The Basic Protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

NASA Astrophysics Data System (ADS)

Chang, Lingqian

Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation techniques. Cells were patterned on the nanochannel array and collectively were electroporated in parallel, injected with cargo in Z-direction. Controlling the dose was demonstrated with the external pulse durations at high-throughput. The 'electrophoretic'- expedited delivery of large molecular weight plasmids were demonstrated with large numbers of primary cells simultaneously, which cannot be achieved in BEP and MEP. Two clinically valuable case studies were performed with our 3D NEP for living cell sensing / interrogation. (1) In the case of in vitro transfection of primary cardiomyocytes, we studied the dose-effects of miR-29 on mitochondrial changes and the suppression of the Mcl-1 gene in adult mouse cardiomyocytes by precisely controlling the miR-29 dose injected. (2) Glioma stem cells (GSCs), a type of cell hypothesized to be highly aggressive and to lead to the relapses of gliobastoma in human brain, was studied at single cell resolution on 3D NEP platform. The developed 3D NEP system moves towards clinically oriented and user-friendly tools for life science applications. The batch-treated cells with controlled dosage delivery provide a useful tool for single cell analysis. The pioneering experiments in this work have demonstrated the 3D NEP for the applications of cell reprogramming, adoptive immunotherapy, in vitro cardiomyocytes transfection and glioma stem cells study.

Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

PubMed

Ding, Jin; Feng, Yuan; Zheng, Zhao Hui; Li, Xue Yi; Wu, Zhen Biao; Zhu, Ping

2015-02-01

Human leucocyte antigen (HLA)-B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA-B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA-B27, EGFP/HLA-A2 or EGFP alone were created to further investigate the relation between HLA-B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA-B27 or β2-microglobulin expression. HLA-B27-transfected U937 cells expressed higher FHC levels than either EGFP/HLA-A2- or EGFP-transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA-B27-transfected cells, implying that FHC, perhaps mostly derived from HLA-B27, plays an important role in SpA pathogenesis. © 2014 John Wiley & Sons Ltd.

[miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

PubMed

Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P <0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P <0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P <0.05) but higher than the miR-143 mimic group significantly ( P <0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the miR-143 mimic+ K-ras group were lowest, moderate, and highest, respectively (115.27±34.08, 521.36±41.89, and 706.52±89.44, all P <0.05). In the tissue samples, the miR-143 expression in the cervical cancer group was significantly lower than that of the cervical intraepithelial neoplasia group (0.32±0.06 vs. 0.93±0.17, P <0.05); whereas the K-ras protein expression in the cervical cancer group was significantly higher than that in the cervical intraepithelial neoplasia group (584.39±72.34 vs. 114.23±25.82, P <0.05). Conclusions: In vitro, miR-143 can inhibit the proliferative activity of HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

Fluorescent carbohydrate probes for cell lectins

NASA Astrophysics Data System (ADS)

Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

2001-09-01

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells

PubMed Central

Xu, Lin-Feng; Ni, Jia-Yan; Sun, Hong-Liang; Chen, Yao-Ting; Wu, Yu-Dan

2013-01-01

AIM: To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells. METHODS: The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM, and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay. RESULTS: Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group; in the siRNA transfection group, the amount of hepatoma cells in G1 phase was more than that in the control group. The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group. The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells. CONCLUSION: Hypoxia increases the expression of HIF-1α, and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells. PMID:23555163

Chemical Oxidation of a Redox-Active, Ferrocene-Containing Cationic Lipid: Influence on Interactions with DNA and Characterization in the Context of Cell Transfection

PubMed Central

Aytar, Burcu S.; Muller, John P. E.; Golan, Sharon; Kondo, Yukishige; Talmon, Yeshayahu; Abbott, Nicholas L.; Lynn, David M.

2012-01-01

We report an approach to the chemical oxidation of a ferrocene-containing cationic lipid [bis(11-ferrocenylundecyl)dimethylammonium bromide, BFDMA] that provides redox-based control over the delivery of DNA to cells. We demonstrate that BFDMA can be oxidized rapidly and quantitatively by treatment with Fe(III)sulfate. This chemical approach, while offering practical advantages compared to electrochemical methods used in past studies, was found to yield BFDMA/DNA lipoplexes that behave differently in the context of cell transfection from lipoplexes formed using electrochemically oxidized BFDMA. Specifically, while lipoplexes of the latter do not transfect cells efficiently, lipoplexes of chemically oxidized BFDMA promoted high levels of transgene expression (similar to levels promoted by reduced BFDMA). Characterization by SANS and cryo-TEM revealed lipoplexes of chemically and electrochemically oxidized BFDMA to both have amorphous nanostructures, but these lipoplexes differed significantly in size and zeta potential. Our results suggest that differences in zeta potential arise from the presence of residual Fe2+ and Fe3+ ions in samples of chemically oxidized BFDMA. Addition of the iron chelating agent EDTA to solutions of chemically oxidized BFDMA produced samples functionally similar to electrochemically oxidized BFDMA. These EDTA-treated samples could also be chemically reduced by treatment with ascorbic acid to produce samples of reduced BFDMA that do promote transfection. Our results demonstrate that entirely chemical approaches to oxidation and reduction can be used to achieve redox-based ‘on/off’ control of cell transfection similar to that achieved using electrochemical methods. PMID:22980739

Ergosterol Peroxide Isolated from Ganoderma lucidum Abolishes MicroRNA miR-378-Mediated Tumor Cells on Chemoresistance

PubMed Central

Li, Xiang-Min; Yang, Weining; Jiao, Chun-Wei; Fang, Ling; Li, Sen-Zhu; Pan, Hong-Hui; Yee, Albert J.; Lee, Daniel Y.; Li, Chong; Zhang, Zhi; Guo, Jun; Yang, Burton B.

2012-01-01

Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells. PMID:22952996

Humanized Androgen Receptor Mice: A Genetic Model for Differential Response to Prostate Cancer Therapy

DTIC Science & Technology

2010-05-01

450 ng PSA reporter, 50 ng renilla internal control, 25 ng receptor alone (12Q or 21Q AR), and with either 25 ng of (ca) Raf1-Kinase or pCMV5 empty...additional 24 hours. The graphs represent relative luciferase activities normalized to renilla for 3 independent trials. Fold activation is calculated as...cells were transfected with 400 ng 3XHRE3 reporter, 100 ng renilla internal control, and 4 ng receptor. 24 hours post transfection cells were fed with

Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering.

PubMed

O'Brien, Paul J; Elahipanah, Sina; Rogozhnikov, Dmitry; Yousaf, Muhammad N

2017-05-24

The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types. Moreover the transfection is efficient with high cell viability and does not require a postsorting step to separate transfected from nontransfected cells in the cell population. We also show for the first time a precision transfection strategy where a single cell type in a coculture is target transfected via bio-orthogonal click chemistry.

Identification of targets of miRNA-221 and miRNA-222 in fulvestrant-resistant breast cancer

PubMed Central

Liu, Pengfei; Sun, Manna; Jiang, Wenhua; Zhao, Jinkun; Liang, Chunyong; Zhang, Huilai

2016-01-01

The present study aimed to identify the differentially expressed genes (DEGs) regulated by microRNA (miRNA)-221 and miRNA-222 that are associated with the resistance of breast cancer to fulvestrant. The GSE19777 transcription profile was downloaded from the Gene Expression Omnibus database, and includes data from three samples of antisense miRNA-221-transfected fulvestrant-resistant MCF7-FR breast cancer cells, three samples of antisense miRNA-222-transfected fulvestrant-resistant MCF7-FR cells and three samples of control inhibitor (green fluorescent protein)-treated fulvestrant-resistant MCF7-FR cells. The linear models for microarray data package in R/Bioconductor was employed to screen for DEGs in the miRNA-transfected cells, and the pheatmap package in R was used to perform two-way clustering. Pathway enrichment was conducted using the Gene Set Enrichment Analysis tool. Furthermore, a miRNA-messenger (m) RNA regulatory network depicting interactions between miRNA-targeted upregulated DEGs was constructed and visualized using Cytoscape. In total, 492 and 404 DEGs were identified for the antisense miRNA-221-transfected MCF7-FR cells and the antisense miRNA-222-transfected MCF7-FR cells, respectively. Genes of the pentose phosphate pathway (PPP) were significantly enriched in the antisense miRNA-221-transfected MCF7-FR cells. In addition, components of the Wnt signaling pathway and cell adhesion molecules (CAMs) were significantly enriched in the antisense miRNA-222-transfected MCF7-FR cells. In the miRNA-mRNA regulatory network, miRNA-222 was demonstrated to target protocadherin 10 (PCDH10). The results of the present study suggested that the PPP and Wnt signaling pathways, as well as CAMs and PCDH10, may be associated with the resistance of breast cancer to fulvestrant. PMID:27895744

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

PubMed

Song, Linyang; Song, Wei; Schipper, Hyman M

2007-08-01

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.

PubMed

Hornstein, Benjamin D; Roman, Dany; Arévalo-Soliz, Lirio M; Engevik, Melinda A; Zechiedrich, Lynn

2016-01-01

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells

PubMed Central

Hornstein, Benjamin D.; Roman, Dany; Arévalo-Soliz, Lirio M.; Engevik, Melinda A.

2016-01-01

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery. PMID:27918590

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

NASA Astrophysics Data System (ADS)

Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

2016-04-01

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro transfection results from BCPV-siRNA, a newly developed biodegradable transfection agent, BCPV, is being probed for transfection performance in an animal model.

Georgetown Institute for Cognitive and Computational Sciences

DTIC Science & Technology

2000-03-01

in neuronal apoptosis. Cerebellar granules cells (CGCs) were co-transfected with a green fluorescent protein reporter and one of several hammerhead ... ribozymes constructed to cleave caspase-3 RNA. Use of such ribozymes is highly selective. In separate experiments we co-transfected with a gene that...expressing a ribozyme against rat caspase-3. Apoptosis was assessed after 24, 36, or 48 h of serum/K+ deprivation. In negative control cells expressing ß

MicroRNA Inhibitors as Anticancer Therapies

DTIC Science & Technology

2007-08-17

Promoter activity was determined by co-transfection of the pGL3 promoter reporter (400 ng/well) with pRLSV40 ( Renilla luciferase, Promega)(100 ng/well) into...performed in triplicate and standard deviations calculated. Activitywas defined as Firefly/ Renilla ratio, normalized to control vector transfection. For...was defined as Firefly/ Renilla ratio normalized to activity in the absence of transfected E2F1. 5-RACE Mapping of Transcript—HEK-293 cells were tran

Transfection using DEAE-dextran.

PubMed

Selden, R F

2001-05-01

Two protocols for DEAE-dextran transfection of cells are provided in this unit. The Basic Protocol describes a procedure used to transfect adherent cells and the first Alternate Protocol presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second Alternate Protocol.

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

PubMed Central

Hunt, Michelle A.; Currie, Margaret J.; Robinson, Bridget A.; Dachs, Gabi U.

2010-01-01

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays. PMID:20592869

[Transfection of hBcl-2 gene protects the liver against ischemia/reperfusion injury in rats during liver transplantation].

PubMed

Liu, Ji-tong; Liu, Jing-shi; Jiang, Jin-yu; Zhou, Li-xue; Liang, Gang; Li, Yan-chun

2010-12-01

To study the effect of hBcl-2 gene transfer on rat liver against ischemia-reperfusion injury, and explore the feasibility of this approach to reduce ischemia-reperfusion injury in liver transplantation. We constructed the replication-deficient recombinant adenoviruses Adv-EGFP and Adv-Bcl-2 and transfected them into 293 cells and packaged into adenovirus particles for amplification and purification. The empty plasmid vector virus was constructed similarly. Male SD rats were randomized into Adv-Bcl-2-transfected group, Adv-EGFP-transfected group, ischemia-reperfusion group, and sham-operated group, and liver allograft transplantation model was established by sleeve method. In the transfected groups, the recombinant viruses were administered by perfusion through the portal vein, and the ischemia-reperfusion and sham-operated groups received no treatment. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expressions of bcl-2 in the liver tissue of each group, and at 0, 60 and 180 min after reperfusion, serum AST, LDH, and MDA levels were measured. Histological changes of the liver cells were evaluated by HE staining. Bcl-2 mRNA and protein expressions in Adv-Bcl-2-transfected group, as compared with those in Adv-EGFP-transfected group and control group, were significantly increased (P<0.01); the serum levels of AST, LDH and MDA in Adv-Bcl-2-transfected group were significantly lower than those of Adv-EGFP-transfected group and ischemia-reperfusion group (P<0.05 or 0.01). Compared with the sham-operated group, Adv-Bcl-2 treatment group showed lessened edema and vacuolar degeneration of the liver cells without patches or spots of necrosis. In ischemia-reperfusion and Adv-EGFP group, HE staining revealed hepatic lobular destruction and extensive liver cell swelling, enlargement, vacuolar degeneration, edema and occasional focal necrosis. Adv-Bcl-2 transfection can induce the expression of bcl-2 gene to reduce ischemia-reperfusion injury of the liver graft in rats.

Alteration in gene expression profile and oncogenicity of esophageal squamous cell carcinoma by RIZ1 upregulation.

PubMed

Dong, Shang-Wen; Li, Dong; Xu, Cong; Sun, Pei; Wang, Yuan-Guo; Zhang, Peng

2013-10-07

To investigate the effect of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) upregulation in gene expression profile and oncogenicity of human esophageal squamous cell carcinoma (ESCC) cell line TE13. TE13 cells were transfected with pcDNA3.1(+)/RIZ1 and pcDNA3.1(+). Changes in gene expression profile were screened and the microarray results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Nude mice were inoculated with TE13 cells to establish ESCC xenografts. After two weeks, the inoculated mice were randomly divided into three groups. Tumors were injected with normal saline, transfection reagent pcDNA3.1(+) and transfection reagent pcDNA3.1(+)/RIZ1, respectively. Tumor development was quantified, and changes in gene expression of RIZ1 transfected tumors were detected by RT-PCR and Western blotting. DNA microarray data showed that RIZ1 transfection induced widespread changes in gene expression profile of cell line TE13, with 960 genes upregulated and 1163 downregulated. Treatment of tumor xenografts with RIZ1 recombinant plasmid significantly inhibited tumor growth, decreased tumor size, and increased expression of RIZ1 mRNA compared to control groups. The changes in gene expression profile were also observed in vivo after RIZ1 transfection. Most of the differentially expressed genes were associated with cell development, supervision of viral replication, lymphocyte costimulatory and immune system development in esophageal cells. RIZ1 gene may be involved in multiple cancer pathways, such as cytokine receptor interaction and transforming growth factor beta signaling. The development and progression of esophageal cancer are related to the inactivation of RIZ1. Virus infection may also be an important factor.

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

PubMed

Wang, Guang; Chen, En-Ni; Liang, Chang; Liang, Jianxin; Gao, Lin-Rui; Chuai, Manli; Münsterberg, Andrea; Bao, Yongping; Cao, Liu; Yang, Xuesong

2018-04-01

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7 + neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7 + and HNK-1 + cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1 + neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU + neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

The regulated in development and DNA damage response 2 (REDD2) gene mediates human monocyte cell death through a reduction in thioredoxin-1 expression.

PubMed

Imen, Jguirim-Souissi; Billiet, Ludivine; Cuaz-Pérolin, Clarisse; Michaud, Nadège; Rouis, Mustapha

2009-05-15

In a previous study, we identified the regulated in development and DNA damage response 2 (REDD2) gene as a highly expressed gene in human atherosclerotic lesions in comparison to normal artery, as well as in cultured human macrophages, and showed its implication in oxidized low-density lipoprotein (LDL)-induced macrophage death sensitivity. In this article, we attempt to identify the mechanism by which REDD2 induces such a phenomenon. Transient transfection of U-937 monocytic cells with a pCI.CMV.REDD2 expression vector increased by approximately twofold the mRNA levels of REDD2 in comparison to control cells transfected with pCI.CMV.GFP. Reactive oxygen species (ROS) production was significantly induced in REDD2-transfected cells compared with control cells (157+/-48 and 100+/-8 arbitrary units/mg cell protein, respectively; p<0.05). Moreover, a significant increase in parameters known to reflect the oxidative modifications of LDL was observed. Among enzymes involved in ROS production or degradation, we found a specific reduction in thioredoxin-1 (Trx-1) mRNA ( approximately 52+/-7% decrease, p<0.01 vs control cells) and protein ( approximately 60+/-4% decrease, p<0.001 vs control cells) levels in cells overexpressing REDD2 in comparison to control cells. In contrast, transfection of U-937 cells with siRNA against REDD2 decreased the mRNA levels of REDD2 by approximately 60% and increased Trx-1 mRNA and protein levels. Moreover, we observed no or a moderate increase in Bax (proapoptotic) and a significant decrease in Bcl2 (antiapoptotic) gene expression in cells that overexpress REDD2 compared to control cells. In addition, we showed that Trx-1 mRNA and protein levels were increased at low H(2)O(2) doses and decreased at higher doses. Interestingly, macrophages isolated from human atherosclerotic lesions differentially express REDD2 and Trx-1. Indeed, in certain patients, levels of REDD2 mRNA were low and those of Trx-1 mRNA were high. In contrast, in other patients, levels of REDD2 were high and levels of Trx-1 mRNA were low.

Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase.

PubMed

Xie, Ying; Zhao, Xiaoe; Jia, Hongxiang; Ma, Baohua

2013-01-01

Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened.

Enhancement of periodontal tissue regeneration by transplantation of osteoprotegerin-engineered periodontal ligament stem cells.

PubMed

Su, Fang; Liu, Shi-Sen; Ma, Jun-Li; Wang, Dong-Sheng; E, Ling-Ling; Liu, Hong-Chen

2015-03-12

The objective of the present study was to evaluate the capacity of a tissue-engineered complex of human osteoprotegerin (hOPG)-transfected periodontal ligament stem cells (PDLSCs) seeding on beta-tricalcium phosphate (β-TCP) to regenerate alveolar bone defects in New Zealand rabbits. PDLSCs were isolated from rabbit periodontal ligament tissues and expanded in vitro to enrich PDLSC numbers, and their proliferative activities and differentiation capability were evaluated under specific induction conditions. Lentiviral vector containing hOPG and enhanced green fluorescent protein (EGFP) was constructed by using Gateway technology and transfected into rabbit PDLSCs. The expression of hOPG was determined with quantitative real-time reverse transcription-polymerase chain reaction and Western blot. The PDLSCs with or without engineered hOPG were seeded on β-TCP scaffolds prior to transplantation. Morphological characterization of cells and materials was done by scanning electron microscope. Twenty rabbits with alveolar bone defects were randomly allocated into four groups and transplanted with β-TCP, PDLSCs/β-TCP, and hOPG-transfected PDLSCs/β-TCP or were left untreated as a control. Animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis. PDLSCs expressed STRO-1 and vementin and favored osteogenesis and adipogenesis in conditioned media. Expressions of hOPG were significantly upregulated after transfection of the lentiviral vector into PDLSCs. PDLSCs attached and spread well on β-TCP, and there was no significant difference in growth of PDLSCs on β-TCP between the hOPG transfection group and the non-transfection group. The histological observation and histomorphometric analysis showed that the hOPG-transfected PDLSCs/β-TCP complex exhibited an earlier mineralization and more bone formation inside the scaffold than control, β-TCP, and PDLSCs/β-TCP complexes. Implantation of hOPG-transfected PDLSCs contributed to new bone formation as determined by EGFP gene expression under circularly polarized light microscopy. The present study demonstrated the feasibility of β-TCP scaffolds for primary PDLSC culture and expression of hOPG gene in vitro and in vivo, and hOPG-transfected PDLSCs could serve as a potential cell source for periodontal bone regeneration, which may shed light on the potential of systemic hOPG gene therapy in combination with PDLSC tissue engineering as a good candidate in periodontal tissue engineering for alveolar bone regeneration.

[The transfection and expression of IL-1ra gene to the rabbit cornea in situ via cation polymer mediation].

PubMed

Yuan, Jin; Chen, Jia-qi; Zhou, Shi-you; Liu, Zu-guo; Wang, Zhi-chong; Gu, Jian-jun

2006-08-01

To investigate the efficiency and safety of transfection of PEGFP-IL-1ra plasmid via cation polymer mediation (poly-ethylenimine, PEI) by injection into the corneal stroma. Human IL-1ra cDNA fragments were cloned by RT-PCR. Plasmid PEGFP-hIL-1ra recombinants were constructed and transferred into corneal endothelial cells (CEC) via cation polymer mediation. Expression of IL-1ra mRNA and IL-1ra was detected by green fluorescent protein (GFP) and Western-blotting. In the experiment group, 20 microl preparation containing 10 microg plasmid PEGFP-hIL-1ra recombinants and PEI-in-vivo was injected into the corneal stroma of Wistar rats (n = 30). Equivalent PEI-in-vivo solution was injected into another 15 corneas as the controls. Corneas were harvested at different time points (day 1, 3, 6, 14 and 21) after injection. The changes of tissue structure and function after IL-1ra in situ transfection were studied by HE staining, transmission electron microscopy, trypan blue-alizarin red staining and immunohistochemistry. The location and intensity of IL-1ra-GFP fusion protein expression were monitored by fluorescence microscopy. The size of the RT-PCR product of hIL-1ra fragments was approximately 500 bp in agarose gel electrophoresis. Restrictive enzyme digestion analysis of PstI, BamHI and DNA sequence analysis showed that expression of plasmid PEGFP-hIL-1ra recombinants had been constructed successfully. Twelve hours after the transfection of PEGFP-hIL-1ra, GFP fluorescence was detected in 10% - 15% endothelial cells. IL-1ra protein (RMW: 44,000) was detected by Western-blotting. In PEGFP-hIL-1ra treated group, fluorescence was appeared at day 1 in cornea basal epithelial cells, peaked at day 6 in whole cornea, began to weaken at day 14, and only weak fluorescence remained in cornea epithelial cells at day 21. No fluorescence appeared in the control group. No significant pathologic changes could be found in HE stained cornea tissues in both transfected group and the controls. p63 immunocytochemical staining in cornea epithelium was positive in both groups. Trypan blue-alizarin red staining confirmed that there was no damage in cornea endothelial cells. IL-1ra-GFP granules could be found by transmission electron microscope in every layer of cornea in the transfected group, but none in the controls. There was no impairment in the ultrastructure of cells in both groups. By direct injection of PEGFP-hIL-1ra into corneal stroma and mediated by cation polymer, IL-1ra genes could be transferred and expressed in corneal tissue efficiently and safely, and might provide a novel technique of gene transfection to cornea in situ.

The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.

PubMed

Jiwaji, Meesbah; Daly, Rónán; Pansare, Kshama; McLean, Pauline; Yang, Jingli; Kolch, Walter; Pitt, Andrew R

2010-12-31

The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid. The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes. Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data.

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

PubMed

Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

2015-09-15

Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. Copyright © 2015 Elsevier Inc. All rights reserved.

Accumulation of a soluble form of human nectin-2 is required for exerting the resistance against herpes simplex virus type 2 infection in transfected cells.

PubMed

Fujimoto, Y; Ozaki, K; Iwamori, N; Takakuwa, H; Ono, E

2016-03-01

Cell entry of herpes simplex virus type 2 (HSV-2) requires the interaction of viral glycoprotein D (gD) with the receptor nectin-1 and herpesvirus entry mediator (HVEM). In addition, it is known that nectin-2 is also functional as a receptor for HSV-2, although the binding to the gD is weak. To examine an antiviral potential of a soluble form of human nectin-2 (hNectin-2Ig), transfected Vero cells expressing the entire ectodomain of nectin-2 fused to the Fc portion of human IgG were established. Specific binding of hNectin-2Ig to HSV-2 gD was confirmed by ELISA. Competitive ELISA demonstrated that accumulation of hNectin-2Ig in transfected cells increased significantly in a cell culture time dependent manner. Viral growth of several HSV-2 strains was significantly inhibited in the transfected cells that were cultured for 72 hr compared with control Vero cells, but not in cells that were cultured for 24 hr. These results indicate that accumulation of a soluble form of nectin-2 is required for exerting the resistance against HSV-2 infection.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

PubMed

Hu, X Y; Fang, Q; Ma, D; Jiang, L; Yang, Y; Sun, J; Yang, C; Wang, J S

2016-06-10

Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

Over-expression of angiotensin II type 2 receptor gene induces cell death in lung adenocarcinoma cells.

PubMed

Pickel, Lara; Matsuzuka, Takaya; Doi, Chiyo; Ayuzawa, Rie; Maurya, Dharmendra Kumar; Xie, Sheng-Xue; Berkland, Cory; Tamura, Masaaki

2010-02-01

The endogenous angiotensin II (Ang II) type 2 receptor (AT 2) has been shown to mediate apoptosis in cardiovascular tissues. Thus, the aim of this study was to explore the anti-cancer effect of AT 2 over-expression on lung adenocarcinoma cells in vitro using adenoviral (Ad), FuGENE, and nanoparticle vectors. All three gene transfection methods efficiently transfected AT 2 cDNA into lung cancer cells but caused minimal gene transfection in normal lung epithelial cells. Ad-AT 2 significantly attenuated multiple human lung cancer cell growth (A549 and H358) as compared to the control viral vector, Ad-LacZ, when cell viability was examined by direct cell count. Examination of annexin V by flow cytometry revealed the activation of the apoptotic pathway via AT 2 over-expression. Western Blot analysis confirmed the activation of caspase-3. Similarly, poly (lactide-co-glycolic acid) (PLGA) biodegradable nanoparticles encapsulated AT 2 plasmid DNA were shown to be effectively taken up into the lung cancer cell. Nanoparticle-based AT 2 gene transfection markedly increased AT 2 expression and resultant cell death in A549 cells. These results indicate that AT 2 over-expression effectively attenuates growth of lung adenocarcinoma cells through intrinsic apoptosis. Our results also suggest that PLGA nanoparticles can be used as an efficient gene delivery vector for lung adenocarcinoma targeted therapy.

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection.

PubMed

Van Pham, Phuc; Thi-My Nguyen, Phuoc; Thai-Quynh Nguyen, Anh; Minh Pham, Vuong; Nguyen-Tu Bui, Anh; Thi-Tung Dang, Loan; Gia Nguyen, Khue; Kim Phan, Ngoc

2014-06-01

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

PubMed

Molenda, Natalia; Urbanova, Katarina; Weiser, Nelly; Kusche-Vihrog, Kristina; Günzel, Dorothee; Schillers, Hermann

2014-01-01

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.

Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation

PubMed Central

Devalliere, Julie; Chang, William G.; Andrejecsk, Jillian W.; Abrahimi, Parwiz; Cheng, Christopher J.; Jane-wit, Dan; Saltzman, W. Mark; Pober, Jordan S.

2014-01-01

Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin αvβ3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (≥6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.—Devalliere, J., Chang, W. G., Andrejecsk, J. W., Abrahimi, P., Cheng, C. J., Jane-wit, D., Saltzman, W. M., Pober, J. S. Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation. PMID:24221087

Betacellulin overexpression in mesenchymal stem cells induces insulin secretion in vitro and ameliorates streptozotocin-induced hyperglycemia in rats.

PubMed

Paz, Ana H; Salton, Gabrielle Dias; Ayala-Lugo, Ana; Gomes, Cristiano; Terraciano, Paula; Scalco, Rosana; Laurino, Claudia Cilene Fernandes Correia; Passos, Eduardo Pandolfi; Schneider, Marlon R; Meurer, Luise; Cirne-Lima, Elizabeth

2011-02-01

Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic β-cells and to improve glucose metabolism in experimental diabetic rodent models. Mesenchymal stem cells (MSCs) have been already proved to be multipotent. Recent work has attributed to rat and human MSCs the potential to differentiate into insulin-secreting cells. Our goal was to transfect rat MSCs with a plasmid containing BTC cDNA to guide MSC differentiation into insulin-producing cells. Prior to induction of cell MSC transfection, MSCs were characterized by flow cytometry and the ability to in vitro differentiate into mesoderm cell types was evaluated. After rat MSC characterization, these cells were electroporated with a plasmid containing BTC cDNA. Transfected cells were cultivated in Dulbecco's modified Eagle medium high glucose (H-DMEM) with 10 mM nicotinamide. Then, the capability of MSC-BTC to produce insulin in vitro and in vivo was evaluated. It was possible to demonstrate by radioimmunoassay analysis that 10(4) MSC-BTC cells produced up to 0.4 ng/mL of insulin, whereas MSCs transfected with the empty vector (negative control) produced no detectable insulin levels. Moreover, MSC-BTC were positive for insulin in immunohistochemistry assay. In parallel, the expression of pancreatic marker genes was demonstrated by molecular analysis of MSC-BTC. Further, when MSC-BTC were transplanted to streptozotocin diabetic rats, BTC-transfected cells ameliorated hyperglycemia from over 500 to about 200 mg/dL at 35 days post-cell transplantation. In this way, our results clearly demonstrate that BTC overabundance enhances glucose-induced insulin secretion in MSCs in vitro as well as in vivo.

Novel Therapeutic and Prophylactic Modalities to Protect U.S. Armed Forces Against Major Biological Threat Agents

DTIC Science & Technology

2004-10-01

using flow cytometry after staining with CBA kit produced by BD-Pharmingen. The CBA kit can simultaneously test 5 inflammatory cytokines that include...or TLR4 transfected cells using flow cytometry . CHOK1 and CHOR1.1 cells were plated out in a 24-well dish and transfected 24 h later with either TLR2...with PE-labeled anti-hTLR2 antibody or PE-isotype control antibody (eBiosciences, CA), and cells were analyzed by flow cytometry . 39 The expression

Selective Gene Regulation by Androgen Receptor in Prostate Cancer

DTIC Science & Technology

2012-10-01

empty vector, wt AR, AR-E255K and AR- R753Q cells were transfected with an ARE- responsive reporter and renilla as control. Cells were treated with...empty vector (empty), wild-type AR (WT), AR-E255K or AR-R753Q were transfected with ARE-luciferase and renilla . Cells were treated with 0 or 1 nm...R1881, harvested after 24 hrs to read luciferase and renilla actiivity. % G ro w th (D ay 5 / D ay 1 ) Vector WTAR E255KR753Q 600 700 800 900

Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

NASA Astrophysics Data System (ADS)

Vernon, Matthew Martin

Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation.

PubMed

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C; Miles, Gareth B; Dholakia, Kishan; Gunn-Moore, Frank J

2013-11-21

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided "point-and-transfect" user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.

Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.

PubMed

Martinho, F C; Camargo, S E A; Fernandes, A M M; Campos, M S; Prado, R F; Camargo, C H R; Valera, M C

2018-01-01

To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers. Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level. The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P < 0.05) and exhibited the highest inflammatory potential against the SV40 T-Ag-transfected cells (P < 0.05). All root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P < 0.05). Higher levels of IL-6, IL-8 and TNF-α were found in cell supernatant after stimulation with multimethacrylate (Real Seal) compared to all other sealers tested (P < 0.05). No differences were found comparing epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05). A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against immortalized pulp cells compared to an epoxy resin-based sealer (AH Plus), a methacrylate-based sealer (EndoRez) and a calcium hydroxide-based sealer (Apexit). © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Toward Contactless Biology: Acoustophoretic DNA Transfection

NASA Astrophysics Data System (ADS)

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-02-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Toward Contactless Biology: Acoustophoretic DNA Transfection.

PubMed

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-02-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Toward Contactless Biology: Acoustophoretic DNA Transfection

PubMed Central

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-01-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors. PMID:26828312

[Expression of SLP-2 mRNA in endometrial cancer and its significance].

PubMed

Feng, Wang-qin; Cui, Zhu-mei; Feng, Feng-zhi; Qi, Xiu-juan; Ding, Fang; Li, Wen-dong; Liu, Zhi-hua

2005-08-01

To characterize the differential expression of SLP-2 in endometrial cancer, and to study the effect of human SLP-2 gene on human endometrial cancer cell line. The expression of SLP-2 gene in 32 cases of endometrial cancer and 28 cases of normal endometrial tissues was examined by semi-quantitative RT-PCR. Eukaryotic expression vectors of sense and antisense SLP-2 were constructed and transfected into HEC-1B cell line using lipofectamine 2000 respectively. The morphological changes of the cell were observed by phase contrast microscopy. The cell growth was detected by methyl thiazolyl tetrazolium (MTT) assay, and the cell cycles were analyzed by flow cytometry. The expression of SLP-2 mRNA in endometrial cancer tissues was higher than that in normal endometrial tissues (1.6 +/- 0.7 vs 0.7 +/- 0.3, P < 0.05). Sense and antisense human SLP-2 constructs were transfected into HEC-1B cell line respectively. After being transfected with sense SLP-2, the expression of SLP-2 mRNA in HEC-1B cell line was increased by about 2.4 times that of the control group, the cell growth was accelerated, and the number of cells in G(1) phase was decreased by 12.5%, S phase was increased by 8.0%. After being transfected with antisense SLP-2, the expression of SLP-2 mRNA was declined 50%. The transfected cells showed slower growth, and the number of cells in G(1) phase was significantly increased by 10.5%, and S phase was declined by 9.8%. SLP-2 mRNA shows up-regulation in endometrial cancer tissues, and it may have some relationship with carcinogenesis of endometrial cancer.

Enhanced p53 gene transfer to human ovarian cancer cells using the cationic nonviral vector, DDC.

PubMed

Kim, Chong-Kook; Choi, Eun-Jeong; Choi, Sung-Hee; Park, Jeong-Sook; Haider, Khawaja Hasnain; Ahn, Woong Shick

2003-08-01

Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo. DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed. The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group. The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.

Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles

PubMed Central

Fan, Z.; Chen, D.; Deng, C.X.

2013-01-01

Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. We conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmid coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9% ± 2.2% (n = 9), comparable with lipofection (7.5% ± 0.8%, n = 9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection. PMID:23770009

Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport

PubMed Central

Kuteykin-Teplyakov, Konstantin; Luna-Tortós, Carlos; Ambroziak, Kamila; Löscher, Wolfgang

2010-01-01

Background and purpose: P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport. Experimental approach: Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine. Key results: In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells. Conclusions and implications: Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport. PMID:20590635

Composition of PLGA and PEI/DNA nanoparticles improves ultrasound-mediated gene delivery in solid tumors in vivo.

PubMed

Chumakova, Olga V; Liopo, Anton V; Andreev, Valery G; Cicenaite, Inga; Evers, B Mark; Chakrabarty, Shilla; Pappas, Todd C; Esenaliev, Rinat O

2008-03-18

The goal of this study was to enhance gene delivery and tumor cell transfection in vivo by using a combination of ultrasonication with complex nanoparticles consisting of two types of nanoparticles: PEI/DNA beta-gal plasmid with highly positive zeta-potential and air-filled poly (lactic-co-glycolic acid) (PLGA) particles (with negative zeta-potential) manufactured in our laboratory. The PLGA/PEI/DNA nanoparticles were a colloid with positive zeta-potential and injected i.v. in nude mice with DU145 human prostate tumors. We found that the combination of PLGA/PEI/DNA nanoparticles with ultrasonication substantially enhanced tumor cell transfection in vivo. The overexpression of beta-gal gene was evaluated histochemically and by Western blot analysis. At least an 8-fold increase of the cell transfection efficacy was obtained in irradiated tumors compared to non-irradiated controls, while little to no cell death was produced by ultrasonication.

An investigation of the effect of antisense RNA gene on bovine leukaemia virus reproduction in cell culture.

PubMed

Murovska, M F; Chernobayeva, L G; Miroshnichenko, O I; Tomsons, V P; Konicheva, V V; Ivanova, S V; Tikhonenko, T I

1992-11-01

A possible approach to control of bovine lymphoproliferative disease caused by bovine leukaemia virus (BLV) may be the development of an "antiviral information immunity" based on the effect of anti-sense RNA (asRNA). A numbers of constructs were obtained, under control of various promotors (herpesvirus thymidine kinase, T-antigen SV40 promoter), carrying as DNA against gene X, the expression product of which is a transactivator of viral transcription from the BLV LTR promotor. As a model system for the analysis of antiviral activity of constructs developed, cloned continuous cell lines of BLV-producing FLK cells were used. The level of BLV expression in cells transfected with the constructs was determined by various parameters. Differences were detected in different clones obtained from non-transfected cells, as well as variation between transfected clones, as measured by reverse transcriptase, competitive radio-immunoassay for BLV p24, the viral particle count on agar membrane, and the tumorigenicity for nude mice. The differences in inhibition of expression of BLV genes and their products may be explained in terms of the site of integration of asDNA and the number of integrated copies.

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

PubMed

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging.

PubMed

Liu, Peng-Cheng; Liu, Kuan; Liu, Jun-Feng; Xia, Kuo; Chen, Li-Yang; Wu, Xing

2016-09-27

The effect of overexpressing the Indian hedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a simulated microgravity environment. An adenovirus plasmid encoding the rabbit IHH gene was constructed in vitro and transfected into rabbit BMSCs. Two large groups were used: conventional cell culture and induction model group and simulated microgravity environment group. Each large group was further divided into blank control group, GFP transfection group, and IHH transfection group. During differentiation induction, the expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins in each group were determined. In the conventional model, the IHH transfection group expressed high levels of cartilage-related factors (Coll2 and ANCN) at the early stage of differentiation induction and expressed high levels of cartilage hypertrophy-related factors (Coll10, annexin 5, and ALP) at the late stage. Under the simulated microgravity environment, the IHH transfection group expressed high levels of cartilage-related factors and low levels of cartilage hypertrophy-related factors at all stages of differentiation induction. Under the simulated microgravity environment, transfection of the IHH gene into BMSCs effectively promoted the generation of cartilage and inhibited cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

[Study of the antitumor activity of alveolar macrophages after transfected human INF-γ gene].

PubMed

Zhou, Fengli; Bi, Xiaogang; Zhang, Tiantuo; Huang, Jing

2011-05-01

Alveolar macrophages (AMs) activated have the antitumor activity. The interferon-γ (INF-γ) is one of the stimulating factors. INF-γ can enhance the immune function of AMs in vitro. The aim of this study is to investigate the effect of human INF-γ gene on the antitumor activity of AMs when transfected into the alveolar macrophages (AMs) from the patients with lung cancer in vitro. AMs obtained by brochoalveolar lavage were separated and transfected by INF-γ gene. RT-PCR and ELISA were applied to determine whether the transfection was successful. The levels of tumor necrosis factor α (TNF-α), nitric oxide (NO) and interleukin-1 (IL-1) produced by AMs and the killing activity of AMs against L1210 cells was detected respectively. Both RT-PCR and ELISA demonstrated that human INF-γ gene had been successfully transfected into AMs. When transfected by human INF-γ gene, the levels of TNF-α, NO and IL-1 produced by AMs from the patients with lung cancer and the killing activity of AMs against L1210 cells were significantly higher than those of the control groups. Human INF-γ gene can enhance the antitumor activity of AMs when transfected into AMs from the patients with lung cancer.

Influence of ribosomal protein L39-L in the drug resistance mechanisms of lacrimal gland adenoid cystic carcinoma cells.

PubMed

Ye, Qing; Ding, Shao-Feng; Wang, Zhi-An; Feng, Jie; Tan, Wen-Bin

2014-01-01

Cancer constitutes a key pressure on public health regardless of the economy state in different countries. As a kind of highly malignant epithelial tumor, lacrimal gland adenoid cystic carcinoma can occur in any part of the body, such as salivary gland, submandibular gland, trachea, lung, breast, skin and lacrimal gland. Chemotherapy is one of the key treatment techniques, but drug resistance, especially MDR, seriously blunts its effects. As an element of the 60S large ribosomal subunit, the ribosomal protein L39-L gene appears to be documented specifically in the human testis and many human cancer samples of different origins. Total RNA of cultured drug-resistant and susceptible lacrimal gland adenoid cystic carcinoma cells was seperated, and real time quantitative RT-PCR were used to reveal transcription differences between amycin resistant and susceptible strains of lacrimal gland adenoid cystic carcinoma cells. Viability assays were used to present the amycin resistance difference in a RPL39-L transfected lacrimal gland adenoid cystic carcinoma cell line as compared to control vector and null-transfected lacrimal gland adenoid cystic carcinoma cell lines. The ribosomal protein L39-L transcription level was 6.5-fold higher in the drug-resistant human lacrimal gland adenoid cystic carcinoma cell line than in the susceptible cell line by quantitative RT-PCR analysis. The ribosomal protein L39-L transfected cells revealed enhanced drug resistance compared to plasmid vector-transfected or null-transfected cells as determined by methyl tritiated thymidine (3H-TdR) incorporation. The ribosomal protein L39-L gene could possibly have influence on the drug resistance mechanism of lacrimal gland adenoid cystic carcinoma cells.

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support.

PubMed

Giri, Shibashish; Bader, Augustinus

2014-09-01

Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. We established a conditional human fetal hepatocytes cell line with mesenchymal characteristics. Thus immortalization of human fetal hepatocytes cell line by telomerase biology offers a great challenge to examine basic biological mechanisms which are directly related to human and best cell source having unlimited population doubling for bioartificial support without any risk of replicative senescence and pathogenic risks.

Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo

PubMed Central

Bhutto, Imran; Handa, James T.; Green, Jordan J.

2012-01-01

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases. PMID:22629417

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

PubMed

Slusser-Nore, Andrea; Larson-Casey, Jennifer L; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H; Sens, Donald A; Dunlevy, Jane R

2016-01-01

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

PubMed Central

Slusser-Nore, Andrea; Larson-Casey, Jennifer L.; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

2016-01-01

Background This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Methods Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. Results It was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Conclusions Tumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA. PMID:26783756

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

PubMed

Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M

2006-10-19

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (P<0.0001). MRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (P<0.0001). However, no difference was observed in the expression of Arp2, Arp3 or other WASPs. A neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (P<0.0001), and reversed the morphological effects of MRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

PubMed

Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

2015-06-01

To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

[The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected hv Ad-Rad50-GFP].

PubMed

Yan, Ruicheng; Huang, Jiancong; Zhu, Ling; Chang, Lihong; Li, Jingjia; Wu, Xifu; Ye, Jin; Zhang, Gehua

2015-12-01

The optimal multiplicity of infection (MOI) of the recombinant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNE1 cell infected by this adenovirus were studied. The biological titer of Ad-Rad50-GFP was measured by end point dilution method. The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve. Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope. The expression f mutant Rad50 in the Ad-Rad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection. The biological titer of Ad-Rad50-GFP was 1.26 x 10¹¹ pfu/ml. CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50. Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection, with the high expression of mutant Rad50, and the efficiency still remained about 70% after 72 hours. Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively. MOI = 50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector Ad-Rad50-GFP.

Calcium phosphate transfection of primary hippocampal neurons.

PubMed

Sun, Miao; Bernard, Laura P; Dibona, Victoria L; Wu, Qian; Zhang, Huaye

2013-11-12

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calpain5 expression is decreased in endometriosis and regulated by HOXA10 in human endometrial cells

PubMed Central

Penna, Ivan; Du, Hongling; Ferriani, Rui; Taylor, Hugh S.

2008-01-01

Calpains have been implicated in the regulation of apoptosis. Here, we identified Calpain5 as a target of HOXA10 transcriptional regulation in endometrial cells as well as its aberrant regulation in endometriosis. Histologically confirmed biopsies of endometriosis were obtained from 20 women. Eutopic endometrium was collected by endometrial biopsy from 30 controls and from the 20 subjects with endometriosis. First trimester decidual samples were obtained from five subjects at the time of pregnancy termination. Immunohistochemistry was used to identify Calpain5 expression. Calpain5 was expressed in endometrial stromal and glandular cells throughout the menstrual cycle and in decidua. Calpain5 protein expression was decreased in both stromal and glandular cells from women with endometriosis compared with that of fertile controls. Human endometrial stromal and epithelial cell lines were transfected with pcDNA/HOXA10, HOXA10 siRNA or respective controls. Quantitative real-time RT–PCR was performed to determine expression of HOXA10 and Calpain5 in each group. Transfection of HESC cells with an HOXA10 expression construct led to increased Calpain5 expression, whereas transfection with siRNA resulted in decreased expression. In conclusion, Calpain5 expression is regulated by HOXA10. Calpain5 expression was decreased in endometriosis likely as a result of decreased HOXA10 expression. Decreased apoptosis in endometrial cells may promote the development of endometriosis through a pathway involving HOXA10, Calpain5 and caspase. PMID:18829447

TRAIL overexpression co-regulated by Egr1 and HRE enhances radiosensitivity of hypoxic A549 cells depending on its apoptosis inducing role.

PubMed

Yang, Yan-Ming; Fang, Fang; Li, Xin; Yu, Lei; Wang, Zhi-Cheng

2017-01-01

Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.

Calcium Phosphate Transfection of Primary Hippocampal Neurons

PubMed Central

DiBona, Victoria L.; Wu, Qian; Zhang, Huaye

2013-01-01

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. PMID:24300106

Femtosecond laser assisted photo-transfection and differentiation of mouse embryonic stem cells

NASA Astrophysics Data System (ADS)

Thobakgale, Lebogang; Manoto, Sello; Ombinda Lemboumba, Satuurnin; Maaza, Malik; Mthunzi-Kufa, Patience

2018-02-01

In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.

Overexpression of secretagogin promotes cell apoptosis and inhibits migration and invasion of human SW480 human colorectal cancer cells.

PubMed

Yang, Xiang-Yi; Liu, Qiao-Rui; Wu, Li-Ming; Zheng, Xu-Lei; Ma, Cong; Na, Ri-Su

2018-05-01

In order to investigate the effect of secretagogin (SCGN) on colorectal cancer (CRC) cells apoptosis, invasion and migration in vitro. Expression of SCGN in CRC tissues and the paired adjacent non-tumorous tissues (n = 36) and four human CRC cell lines (HT29, HCT116, SW480 and SW620) were detected. SW480 cells were transfected with the SCGN overexpression plasmid (eGFP-SCGN), si-SCGN-773, and the corresponding negative controls (NCs). Then, cell-cycle distribution, cell apoptosis, migration, invasion and expression of apoptosis- and metastasis-related proteins were detected. SCGN was significantly downregulated in CRC tissues as compared with the adjacent non-tumorous tissues. The expression of SCGN in HT29 and SW480 cells were lower than those in HT116 and SW620 cells. We transfected SW480 cells with SCGN overexpression plasmid eGFP-SCGN and found the increased cell apoptosis, with cell arresting at G0/G1 phase. SW480 cells with SCGN overexpression showed wider wound width and fewer invaded cells than control and blank cells, with upregulated Bax, cleaved Caspase 3 and E-cadherin, and downregulated Bcl-2 and Vimentin. We also transfected SW480 cells with si-SCGN-773 and found si-SCGN increased cell migration and invasion, but did not affect cell apoptosis and expression of related proteins. We concluded that the overexpression of SCGN in SW480 cells promoted cell apoptosis and inhibited cell migration and invasion. Copyright © 2018. Published by Elsevier Masson SAS.

Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies.

PubMed

Kofron, Michelle D; Laurencin, Cato T

2004-06-01

One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide-co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation

PubMed Central

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C.; Miles, Gareth B.; Dholakia, Kishan; Gunn-Moore, Frank J.

2013-01-01

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits. PMID:24257461

Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.

PubMed

Li, Pu; Zhang, Lei

2015-08-01

The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial cells compared with that of gene transfection alone. Furthermore, significantly enhanced reparative effects were observed in the myocardium of rabbits following treatment with Nkx2.5-or GATA-4-transfected myocardial cell extracellular environment co-cultured BMSCs compared with those treated with untreated BMSCs.

In vivo over-expression of KGF mimic human middle ear cholesteatoma.

PubMed

Yamamoto-Fukuda, Tomomi; Akiyama, Naotaro; Shibata, Yasuaki; Takahashi, Haruo; Ikeda, Tohru; Koji, Takehiko

2015-10-01

We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.

Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay.

PubMed

Osorio, Johan S; Bionaz, Massimo

2017-08-30

Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor β/σ (PPARβ/σ) agonist. The GRT assay was unaffected by poor transfection in MACT cells although the high transfection hampered the possibility of detecting differences between 10 and 100nM of the PPARβ/δ agonist. In MDBK cells, low transfection efficiency (<2.0%) failed to detect any differences with GRT assay. The level of transfection was positively associated with a lower coefficient of variation of GRT data. Overall, our data indicates that results of GRT assays are affected by transfection efficiency and a minimum transfection of 2% is required. Thus, factors such as TR type, TR amount, and DNA plasmid amount need to be optimized for a specific cell type before performing GRT assays. Copyright © 2017 Elsevier B.V. All rights reserved.

An optofluidic approach for gold nanoprobes based-cancer theranostics

NASA Astrophysics Data System (ADS)

Panwar, Nishtha; Song, Peiyi; Yang, Chengbin; Yong, Ken-Tye; Tjin, Swee Chuan

2017-02-01

Suppression of overexpressed gene mutations in cancer cells through RNA interference (RNAi) technique is a therapeutically effective modality for oncogene silencing. In general, transfection agent is needed for siRNA delivery. Also, it is a tedious and time consuming process to analyze the gene transfection using current conventional flow cytometry systems and commercially available transfection kits. Therefore, there are two urgent challenges that we need to address for understanding and real time monitoring the delivery of siRNA to cancer cells more effectively. One, nontoxic, biocompatible and stable non-viral transfection agents need to be developed and investigated for gene delivery in cancer cells. Two, new, portable optofluidic methods need to be engineered for determining the transfection efficiency of the nanoformulation in real time. First, we demonstrate the feasibility of using gold nanorods (AuNRs) as nanoprobes for the delivery of Interleukin-8 (IL-8) siRNA in a pancreatic cancer cell line- MiaPaCa-2. An optimum ratio of 10:1 for the AuNRs-siRNA nanoformulation required for efficient loading has been experimentally determined. Promising transfection rates (≈88%) of the nanoprobe-assisted gene delivery are quantified by flow cytometry and fluorescence imaging, which are higher than the commercial control, Oligofectamine. The excellent gene knockdown performance (over 81%) of the proposed model support in vivo trials for RNAi-based cancer theranostics. In addition to cancer theranostics, our nanoprobe combination can be also applied for disease outbreak monitoring like MERS. Second, we present an optical fiber-integrated microfluidic chip that utilizes simple hydrodynamic and optical setups for miniaturized on-chip flow cytometry. The chip provides a powerful and convenient tool to quantitatively determine the siRNA transfection into cancer cells without using bulky flow cytometer. These studies outline the role of AuNRs as potential non-viral gene delivery vehicles, and their suitability for microfluidics-based lab-on-chip flow cytometry applications.

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

PubMed Central

Kukowska-Latallo, J F; Bielinska, A U; Johnson, J; Spindler, R; Tomalia, D A; Baker, J R

1996-01-01

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines. Images Fig. 1 Fig. 2 Fig. 4 PMID:8643500

Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

PubMed

Lu, Ming-Yu; Li, Zhihong; Hwang, Shiaw-Min; Linju Yen, B; Lee, Gwo-Bin

2015-09-01

This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future.

Inhibition of STAT-3 Results in Radiosensitization of Human Squamous Cell Carcinoma

PubMed Central

Bonner, James A.; Trummell, Hoa Q.; Willey, Christopher D.; Plants, Brian A.; Raisch, Kevin P.

2009-01-01

Background Signal Transducer and Activator of Transcription – 3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein may produce radiosensitization. Methods/Results A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25-31 h as compared to 18-24 h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells. Conclusion A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by inhibition of EGFr. PMID:19616333

A new strategy in the treatment of chemoresistant lung adenocarcinoma via specific siRNA transfection of SRF, E2F1, Survivin, HIF and STAT3.

PubMed

Stoleriu, Mircea Gabriel; Steger, Volker; Mustafi, Migdat; Michaelis, Martin; Cinatl, Jindrich; Schneider, Wilke; Nolte, Andrea; Kurz, Julia; Wendel, Hans Peter; Schlensak, Christian; Walker, Tobias

2014-11-01

According to the actual treatment strategies of lung cancer, the current therapeutic regimen is an individualized, multidisciplinary concept. The development of chemoresistance in the last decade represents the most important obstacle to an effective treatment. In our study, we examined a new therapeutic alternative in the treatment of multiresistant lung adenocarcinoma via siRNA-specific transfection of six crucial molecules involved in lung carcinogenesis [serum response factor(SFR), E2F1, Survivin, hypoxia inducible factor1 (HIF1), HIF2 and signal transducer and activator of transcription (STAT3)]. Three chemoresistant A549 adenocarcinoma cells were cultured under standard conditions at 37°C and 5% CO2. The chemoresistance against Vinflunine, Vinorelbine and Methotrexate was induced artificially. The A549 cells were transfected for 2 h at 37°C with specific siRNA targeting SRF, E2F1, Survivin, HIF1, HIF2 and STAT3 in a non-viral manner. The efficiency of siRNA silencing was evaluated via quantitative real-time polymerase chain reaction, whereas the surviving cells after siRNA transfection as predictor factor for tumoural growth were analysed with a CASY cell counter 3 days after transfection. The response of the chemotherapeutic resistant adenocarcinoma cells after siRNA transfection was concentration-dependent at both 25 and 100 nM. The CASY analysis showed a very effective suppression of adenocarcinoma cells in Vinorelbine, Vinflunine and Methotrexate groups, with significantly better results in comparison with the control group. In our study, we emphasized that siRNA interference might represent a productive platform for further research in order to investigate whether a new regimen in the treatment of multiresistant non-small-cell lung cancer could be established in vivo in the context of a multimodal cancer therapy. © The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease: an in vitro study.

PubMed

Kaur, Savneet; Kumar, T R Santhosh; Uruno, Akira; Sugawara, Akira; Jayakumar, Karunakaran; Kartha, Chandrasekharan Cheranellore

2009-11-01

Recent studies have reported a marked impairment in the number and functions of endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). In view of an important role of eNOS in angiogenesis, in the present study, we evaluated the effects of eNOS gene transfer in ex vivo expanded EPCs isolated from patients with CAD. The expanded EPCs were transfected with mammalian expression vector pcDNA3.1-eNOS containing the full-length human eNOS gene using lipofectamine. About 35-40% of the eNOS-EPCs had higher expression of eNOS as compared to untransfected EPCs. EPCs transfected with pcDNA3.0-EGFP, the plasmid vector expressing green fluorescent protein (GFP) were used as control. The untransfected, GFP-transfected and eNOS-transfected EPCs were compared in terms of important functional attributes of angiogenesis such as proliferation, migration, differentiation and adhesion/integration into tube-like structures in vitro. Functional studies revealed that in the presence of defined growth conditions, compared to the untransfected and GFP-transfected cells, eNOS-EPCs from patients with CAD have a significant increase in [3H] thymidine-labeled DNA (P < 0.01), migration (14.6 +/- 1.8 and 16.5 +/- 1.9 vs. 23.5 +/- 3.4 cells/field, P < 0.01), ability to differentiate into endothelial-like spindle-shaped cells (46 +/- 4.5 and 56.5 +/- 2.1 vs. 93.2 +/- 6.6 cells/field, P < 0.001) and also incorporation into tube-like structures on the matrigel (GFP-EPCs: 21.25 +/- 2.9 vs. GFP-eNOS-EPCs: 34.5 +/- 5.5 cells/field, P < 0.05). We conclude that eNOS gene transfection is a valuable approach to augment angiogenic properties of ex vivo expanded EPCs and eNOS-modified EPCs may offer significant advantages than EPCs alone in terms of their clinical use in patients with myocardial ischemia.

Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.

PubMed

Bruzauskaite, Ieva; Bironaite, Daiva; Bagdonas, Edvardas; Skeberdis, Vytenis Arvydas; Denkovskij, Jaroslav; Tamulevicius, Tomas; Uvarovas, Valentinas; Bernotiene, Eiva

2016-04-30

The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs. hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone. Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores. mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

PubMed

Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

2017-08-22

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

PubMed

Gomaa, Fatma; Garcia, Paulo A; Delaney, Jennifer; Girguis, Peter R; Buie, Cullen R; Edgcomb, Virginia P

2017-09-01

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Non-small cell lung carcinoma therapy using mTOR-siRNA.

PubMed

Matsubara, Hirochika; Sakakibara, Kenji; Kunimitsu, Tamo; Matsuoka, Hiroyasu; Kato, Kaori; Oyachi, Noboru; Dobashi, Yoh; Matsumoto, Masahiko

2012-01-01

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition.

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

PubMed

Ikeda, Ritsuko; Kurokawa, Manae S; Chiba, Shunmei; Yoshikawa, Hideshi; Hashimoto, Takuo; Tadokoro, Mamoru; Suzuki, Noboru

2004-10-01

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

Non-small cell lung carcinoma therapy using mTOR-siRNA

PubMed Central

Matsubara, Hirochika; Sakakibara, Kenji; Kunimitsu, Tamo; Matsuoka, Hiroyasu; Kato, Kaori; Oyachi, Noboru; Dobashi, Yoh; Matsumoto, Masahiko

2012-01-01

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition. PMID:22400071

Skeleton-Controlled pDNA Delivery of Renewable Steroid-Based Cationic Lipids, the Endocytosis Pathway Analysis and Intracellular Localization

PubMed Central

Wang, Zhao; Luo, Ting; Cao, Amin; Sun, Jingjing

2018-01-01

Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of “lipid-raft-mediated endocytosis→lysosome→cell nucleic localization” was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application. PMID:29373505

The silencing effect of miR-30a on ITGA4 gene expression in vitro: an approach for gene therapy.

PubMed

Darzi, Leila; Boshtam, Maryam; Shariati, Laleh; Kouhpayeh, Shirin; Gheibi, Azam; Mirian, Mina; Rahimmanesh, Ilnaz; Khanahmad, Hossein; Tabatabaiefar, Mohammad Amin

2017-12-01

Integrins are adhesion molecules which play crucial roles in cell-cell and cell-extracellular matrix interactions. Very late antigen-4 or α4β1 and lymphocyte Peyer's patch adhesion molecule-1 or α4β7, are key factors in the invasion of tumor cells and metastasis. Based on the previous reports, integrin α4 ( ITGA4 ) is overexpressed in some immune disorders and cancers. Thus, inhibition of ITGA4 could be a therapeutic strategy. In the present study, miR-30a was selected in order to suppress ITGA4 expression. The ITGA4 3' UTR was amplified, cloned in the Z2827-M67-( ITGA4 ) plasmid and named as Z2827-M67/3'UTR. HeLa cells were divided into five groups; (1) untreated without any transfection, (2) mock with Z2827-M67/3'UTR transfection and X-tremeGENE reagent, (3) negative control with Z2827-M67/3'UTR transfection alone, (4) test with miR-30a mimic and Z2827-M67/3'UTR transfection and (5) scramble with miR-30a scramble and Z2827-M67/3'UTR transfection. The MTT assay was performed to evaluate cell survival and cytotoxicity in each group. Real-time RT-PCR was applied for the ITGA4 expression analysis. The findings of this study showed that miR-30a downregulated ITGA4 expression and had no effect on the cell survival. Due to the silencing effect of miR-30a on the ITGA4 gene expression, this agent could be considered as a potential tool for cancer and immune disorders therapy.

The silencing effect of miR-30a on ITGA4 gene expression in vitro: an approach for gene therapy

PubMed Central

Darzi, Leila; Boshtam, Maryam; Shariati, Laleh; Kouhpayeh, Shirin; Gheibi, Azam; Mirian, Mina; Rahimmanesh, Ilnaz; Khanahmad, Hossein; Tabatabaiefar, Mohammad Amin

2017-01-01

Integrins are adhesion molecules which play crucial roles in cell-cell and cell-extracellular matrix interactions. Very late antigen-4 or α4β1 and lymphocyte Peyer’s patch adhesion molecule-1 or α4β7, are key factors in the invasion of tumor cells and metastasis. Based on the previous reports, integrin α4 (ITGA4) is overexpressed in some immune disorders and cancers. Thus, inhibition of ITGA4 could be a therapeutic strategy. In the present study, miR-30a was selected in order to suppress ITGA4 expression. The ITGA4 3' UTR was amplified, cloned in the Z2827-M67-(ITGA4) plasmid and named as Z2827-M67/3'UTR. HeLa cells were divided into five groups; (1) untreated without any transfection, (2) mock with Z2827-M67/3'UTR transfection and X-tremeGENE reagent, (3) negative control with Z2827-M67/3'UTR transfection alone, (4) test with miR-30a mimic and Z2827-M67/3'UTR transfection and (5) scramble with miR-30a scramble and Z2827-M67/3'UTR transfection. The MTT assay was performed to evaluate cell survival and cytotoxicity in each group. Real-time RT-PCR was applied for the ITGA4 expression analysis. The findings of this study showed that miR-30a downregulated ITGA4 expression and had no effect on the cell survival. Due to the silencing effect of miR-30a on the ITGA4 gene expression, this agent could be considered as a potential tool for cancer and immune disorders therapy. PMID:29204174

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

PubMed Central

Weiskirchen, Ralf; Kneifel, Jens; Weiskirchen, Sabine; van de Leur, Eddy; Kunz, Dagmar; Gressner, Axel M

2000-01-01

Background The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. Results With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. Conclusions Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells. PMID:11178102

MicroRNA-190 regulates FOXP2 genes in human gastric cancer.

PubMed

Jia, Wen-Zhuo; Yu, Tao; An, Qi; Yang, Hua; Zhang, Zhu; Liu, Xiao; Xiao, Gang

2016-01-01

To investigate how microRNA-190 (miR-190) regulates FOXP2 genes in gastric cancer (GC) cell line SGC7901. We identified that miR-190 could target FOXP2 genes by using dual luciferase enzyme assay. Precursor fragment transfection of miR-190 was performed with GC cell line SGC7901 and human gastric mucosal cell line GES-1. miR-190 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and FOXP2 protein expression was measured by Western blotting. FOXP2-3'-untranslated region (UTR) in miR-190 transfection group was significantly decreased as compared with other groups. There were no significant differences in fluorescence signals of FOXP2mut-3'-UTR in each group. Therefore, it was assumed that miR-190 can target FOXP2 genes. Through RT-PCR verification, it was observed that the expression level of miR-190 was significantly higher in GC cell line SGC7901 than in human gastric mucosa cell line GES-1 after transfection with miR-190 mimics. The expression level of miR-190 was significantly higher in GES-1 cells than in SGC7901 cells after transfection with miR-190 inhibitors. Western blotting results showed the expression level of FOXP2 was significantly lower in GC cell line SGC7901 than in GES-1 cells. Compared with blank, mimics control, and inhibitors control groups, the miR-190 mimics group showed significantly enhanced proliferation, migration, and invasion abilities, while miR-190 inhibitors group showed decreased abilities toward proliferation, migration, and invasion (P<0.05). The transcription level of miR-190 and the expression level of FOXP2 in tumor tissues and adjacent normal tissues in GC patients were verified to be consistent with those of cell line experiments. Upregulation of miR-190 can lead to downregulation of FOXP2 protein expression. miR-190 may serve as a potential target for GC diagnosis.

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

PubMed Central

Henning, Tobias D; Sutton, Elizabeth J; Kim, Anne; Golovko, Daniel; Horvai, Andrew; Ackerman, Larry; Sennino, Barbara; McDonald, Donald; Lotz, Jeffrey; Daldrup-Link, Heike E

2010-01-01

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different Ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with Ferucarbotran using various protocols: Cells were labeled with 100μg Fe/ml for 4h and 18h and additional samples were cultured for 6 or 12 days after the 18-hour labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared to unlabeled controls by staining with alcian blue and hematoxylin & eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3T was investigated on day 1 and day 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T2-SE and T2*-GE-sequences. Iron uptake was significant for all labeling protocols (p< 0.05). The uptake was highest after transfection with protamine sulfate (25.65 ± 3.96 pg/cell) and lowest at an incubation time of 4h without transfection (3.21 ± 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared to the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4h, inhibition of chondrogenesis was not significant. PMID:19670250

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

PubMed

Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

Silencing the hsp25 Gene Eliminates Migration Capability of the Highly Metastatic Murine 4T1 Breast Adenocarcinoma Cell

PubMed Central

Bausero, Maria A.; Bharti, Ajit; Page, Diana T.; Perez, Kristen D.; Eng, Jason W.-L.; Ordonez, Susana L.; Jantschitsch, Christian; Kindas-Muegge, Ingela; Ciocca, Daniel; Asea, Alexzander

2006-01-01

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration. PMID:16340246

Silencing the hsp25 gene eliminates migration capability of the highly metastatic murine 4T1 breast adenocarcinoma cell.

PubMed

Bausero, Maria A; Bharti, Ajit; Page, Diana T; Perez, Kristen D; Eng, Jason W-L; Ordonez, Susana L; Asea, Edwina E; Jantschitsch, Christian; Kindas-Muegge, Ingela; Ciocca, Daniel; Asea, Alexzander

2006-01-01

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration. Copyright 2006 S. Karger AG, Basel.

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

PubMed Central

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells

PubMed Central

Fahrmayr, C; König, J; Auge, D; Mieth, M; Fromm, MF

2012-01-01

BACKGROUND AND PURPOSE The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. EXPERIMENTAL APPROACH Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. KEY RESULTS Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. CONCLUSIONS AND IMPLICATIONS Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition. PMID:21923755

TGF-β1 gene-engineered mesenchymal stem cells induce rat cartilage regeneration using nonviral gene vector.

PubMed

He, Cai-Xia; Zhang, Tian-Yuan; Miao, Pei-Hong; Hu, Zhong-Jie; Han, Min; Tabata, Yasuhiko; Hu, Yu-Lan; Gao, Jian-Qing

2012-01-01

This study evaluated the potential of utilizing transfected pTGFβ-1 gene-engineered rat mesenchymal stem cells (MSCs) using nonviral vector to promote cartilage regeneration. Pullulan-spermine was used as the nonviral gene vector and gelatin sponge was used as the scaffold. MSCs were engineered with TGF-β1 gene with either the three-dimensional (3D) reverse transfection system or the two-dimensional (2D) conventional transfection system. For the 3D reverse transfection system, pullulan-spermine/pTGF-β1 gene complexes were immobilized to the gelatin sponge, followed by the seeding of MSCs. Pullulan-spermine/pTGF-β1 gene complexes were delivered to MSCs cultured in the plate to perform the 2D conventional transfection system, and then MSCs were seeded to the gelatin sponge. Then, TGF-β1 gene-transfected MSC seeded gelatin sponge was implanted to the full-thickness cartilage defect. Compared with the control group, both groups of TGF-β1 gene-engineered MSCs improved cartilage regeneration through optical observation and histology staining. So, with pullulan-spermine as the nonviral vector, TGF-β1-gene engineered MSCs can induce cartilage regeneration in vivo. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Pim-2 activates API-5 to inhibit the apoptosis of hepatocellular carcinoma cells through NF-kappaB pathway.

PubMed

Ren, Ke; Zhang, Wei; Shi, Yujun; Gong, Jianping

2010-06-01

Pim-2 is proved to be relevant to the tumorigenesis of hepatocellular carcinoma (HCC), but the mechanism is unclear. We studied the relationship among Pim-2, NF-kappaB and API-5. In our experiment, expression level of the three factors and phosphorylation level of API-5, as well as NF-kappaB activity, were detected in HCC tissues and the nontumorous controls. Then Pim-2 gene was transfected into nontumorous liver cells L02, and Pim-2 SiRNA was transfected into hepatoblastoma cell line HepG2. Parthenolide was added as NF-kappaB inhibitor. The same detections as above were repeated in the cells, along with the apoptosis analysis. We found the levels of Pim-2, NF-kappaB and API-5, as well as NF-kappaB activity, were significantly higher in HCC tissues. Pim-2 level was increased in L02 cells after the transfection of Pim-2 gene, but decreased in HepG2 cells after the transfection of Pim-2 SiRNA. The levels of NF-kappaB and API-5, as well as NF-kappaB activity and API-5 phosphorylation level, were in accordance with Pim-2 level, but could be reversed by Parthenolide. Cell apoptosis rates were negatively correlated with API-5 phosphorylation level. Therefore, we infer that Pim-2 could activate API-5 to inhibit the apoptosis of liver cells, and NF-kappaB is the key regulator.

Transfection in perfused microfluidic cell culture devices: A case study.

PubMed

Raimes, William; Rubi, Mathieu; Super, Alexandre; Marques, Marco P C; Veraitch, Farlan; Szita, Nicolas

2017-08-01

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

PubMed

Green, David W; Kim, Eun-Jung; Jung, Han-Sung

2015-09-01

The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.

Myosin light chain kinase controls voltage-dependent calcium channels in vascular smooth muscle.

PubMed

Martinsen, A; Schakman, O; Yerna, X; Dessy, C; Morel, N

2014-07-01

The Ca(2+)-dependent kinase myosin light chain kinase (MLCK) is the activator of smooth muscle contraction. In addition, it has been reported to be involved in Ca(2+) channel regulation in cultured cells, and we previously showed that the MLCK inhibitor ML-7 decreases arginine vasopressin (AVP)-induced Ca(2+) influx in rat aorta. This study was designed to investigate whether MLCK is involved in Ca(2+) regulation in resistance artery smooth muscle cell, which plays a major role in the control of blood pressure. As ML compounds were shown to have off-target effects, MLCK was downregulated by transfection with a small interfering RNA targeting MLCK (MLCK-siRNA) in rat small resistance mesenteric artery (RMA) and in the rat embryonic aortic cell line A7r5. Noradrenaline-induced contraction and Ca(2+) signal were significantly depressed in MLCK-siRNA compared to scramble-siRNA-transfected RMA. Contraction and Ca(2+) signal induced by high KCl and voltage-activated Ca(2+) current were also significantly decreased in MLCK-siRNA-transfected RMA, suggesting that MLCK depletion modifies voltage-operated Ca(2+) channels. KCl- and AVP-induced Ca(2+) signals and voltage-activated Ca(2+) current were decreased in MLCK-depleted A7r5 cells. Eventually, real-time quantitative PCR analysis indicated that in A7r5, MLCK controlled mRNA expression of CaV1.2 (L-type) and CaV3.1 (T-type) voltage-dependent Ca(2+) channels. Our results suggest that MLCK controls the transcription of voltage-dependent Ca(2+) channels in vascular smooth muscle cells.

Adeno-associated virus transformation into the normal miniature pig and the normal guinea pigs cochlea via scala tympani.

PubMed

Shi, Xunbei; Wu, Nan; Zhang, Yue; Guo, Weiwei; Lin, Chang; Yang, Shiming

2017-09-01

To investigate the expression of the miniature pig cochlea after AAV1 transfect into the cochlea via round window membrane (RWM). Twenty miniature pigs are equally divided into four experimental groups. Twelve miniature pigs are equally divided into four control groups. Each pig was transfected with the AAV1 in the experimental group via RWM and each pig was transduced with the artificial perilymph in the control group. The expression of green fluorescent protein (GFP) was observed at 2 weeks, 3 weeks and 4 weeks, respectively. Likewise, AAV1 was delivered into the guinea pigs cochleas using the same method, and the results were compared with that of the miniature pigs. The expression was mainly in the inner hair cells of the miniature pig. The expression of GFP began to appear at 2 weeks, reached the peak at 3 weeks. It also expressed in Hensen's cells, inner pillar cells, outer pillar cells, spiral limbus, and spiral ligament. In the meanwhile, AAV1 was delivered into guinea pig cochlea via the same method, and AAV1 was also expressed in the inner hair cells. But the expression peaked at 2 weeks, and the efficiency of the inner hair cell transfection was higher than that of the pig. AAV1 can be transformed into miniature pig cochlea via scala tympani by the RWM method efficiently.

Successful nucleofection of rat adipose-derived stroma cells with Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe).

PubMed

Fülbier, Angela; Schnabel, Reinhild; Michael, Stefanie; Vogt, Peter M; Strauß, Sarah; Reimers, Kerstin; Radtke, Christine

2014-10-09

Adipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs. ASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni's post hoc test. High initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration. Our study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.

Novel mechanism of gene transfection by low-energy shock wave.

PubMed

Ha, Chang Hoon; Lee, Seok Cheol; Kim, Sunghyen; Chung, Jihwa; Bae, Hasuk; Kwon, Kihwan

2015-08-05

Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

Gene Transfection Method Using Atmospheric Pressure Dielectric-Barrier Discharge Plasmas

NASA Astrophysics Data System (ADS)

Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

2013-09-01

Gene transfection which is the process of deliberately introducing nucleic acids into cells is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure dielectric-barrier discharge (AP-DBD) plasmas. AP-DBD He plasmas are irradiated to the living cell covered with genes. Preliminarily, we use fluorescent dye YOYO-1 instead of the genes and use LIVE/DEAD Stain for cell viability test, and we analyze the transfection efficiency and cell viability under the various conditions. It is clarified that the transfection efficiency is strongly dependence on the plasma irradiation time and cell viability rates is high rates (>90%) regardless of long plasma irradiation time. These results suggest that ROS (Reactive Oxygen Species) and electric field generated by the plasma affect the gene transfection. In addition to this (the plasma irradiation time) dependency, we now investigate the effect of the plasma irradiation under the various conditions.

CD10-bearing fibroblast inhibits matrigel invasive potency of interleukin-1α-producing squamous cell carcinoma by diminishing substance P levels in the tumor microenvironment.

PubMed

Xie, Lining; Moroi, Yoichi; Tsuji, Gaku; Liu, Min; Hayashida, Sayaka; Takahara, Masakazu; Fukagawa, Shuji; Takeuchi, Satoshi; Shan, Baoen; Nakahara, Takeshi; Uchi, Hiroshi; Yokomizo, Takehiko; Furue, Masutaka

2010-12-01

CD10 is a neutral endopeptidase, which cleaves various peptide substrates including substance P. CD10 expression has been detected in peritumoral fibroblasts (Fb) within the invasive area of various cancers such as squamous cell carcinoma (SCC). However, the biological significance of CD10-bearing Fb remains largely unknown. We examined dynamic interactions of Fb with tumorigenic A431 SCC cells or non-tumorigenic HaCaT squamous cells. The SCC and HaCaT cells did not synthesize CD10, while Fb constitutively expressed CD10. When co-cultured, SCC markedly upregulated fibroblastic CD10 expression compared with HaCaT, which was mainly attributable to SCC-derived interleukin-1α (IL-1α). Both SCC and Fb autonomously secreted substance P, which eventually enhanced the invasive capacity of SCC in a matrigel invasion assay by upregulating matrix metalloproteinase (MMP)-1 and MMP-2, but not MMP-9. Transfection of siRNA for CD10 successfully knocked down the CD10 expression in Fb (CD10ND-Fb). In the presence of CD10ND-Fb, substance P levels in supernatants as well as MMP production and the invasive potency of SCC were significantly augmented compared with control scramble RNA-transfected Fb. We also transfected CD10 vector to Fb and found that the matrigel invasive ability of SCC cells was downregulated co-cultured with CD10 vector-transfected Fb rather than empty vector-transfected Fb. In conclusion, the CD10-bearing Fb generated by SCC-derived IL-1 inhibited the invasive capacity of SCC by diminishing the microenvironmental concentration of substance P. © 2010 Japanese Cancer Association.

A systematic study of transfection efficiency and cytotoxicity in HeLa cells using iron oxide nanoparticles prepared with organic and inorganic bases.

PubMed

Calmon, Marilia Freitas; de Souza, Aryane Tofanello; Candido, Natalia Maria; Raposo, Maria Irene Bartolomeu; Taboga, Sebastião; Rahal, Paula; Nery, Jose G

2012-12-01

Magnetic iron oxide nanoparticles (magnetite) (MNPs) were prepared using different organic and inorganic bases. Strong inorganic base (KOH) and organic bases (NH(4)OH and 1,4-diazabicyclo[2.2.2]octane (DABCO)) were used in the syntheses of the MNPs. The MNPs were characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR) and magnetization measurements. MNPs prepared with strong inorganic base yielded an average size of 100 nm, whereas the average size of the MNPs prepared with the organic bases was 150 nm. The main competitive phase for MNPs prepared with the strong inorganic and organic bases was maghemite; however, syntheses with KOH yielded a pure magnetite phase. The transfection study performed with the MNPs revealed that the highest transfection rate was obtained with the MNPs prepared with KOH (74%). The correlation between the magnetic parameters and the transfection ratio without transfection agents indicated that MNPs prepared with KOH were a better vector for possible applications of these MNPs in biomedicine. HeLa cells incubated with MNP-KOH at 10 μg/mL for 24 and 48 h exhibited a decrease in population in comparison with the control cells and it was presumably related to the toxicity of the MNPs. However, the cells incubated with MNP-KOH at 50 and 100 μg/mL presented a very small difference in the viability between the cell populations studied at 24 and 48 h. These data illustrate the viability of HeLa cells treated with MNP-KOH and suggest the potential use of these MNPs in biomedical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Oncogene GAEC1 regulates CAPN10 expression which predicts survival in esophageal squamous cell carcinoma

PubMed Central

Chan, Dessy; Tsoi, Miriam Yuen-Tung; Liu, Christina Di; Chan, Sau-Hing; Law, Simon Ying-Kit; Chan, Kwok-Wah; Chan, Yuen-Piu; Gopalan, Vinod; Lam, Alfred King-Yin; Tang, Johnny Cheuk-On

2013-01-01

AIM: To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance. METHODS: The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses. RESULTS: The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16.33 vs 12.62 ± 12.44, P = 0.032). No significant correction was observed among the TNRC6C protein expression level and the clinocopathologcial features of esophageal squamous cell carcinoma. CONCLUSION: GAEC1 regulates the expression of CAPN10 and TNRC6C downstream. Calpain 10 expression is a potential prognostic marker in patients with esophageal squamous cell carcinoma. PMID:23687414

[Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

PubMed

Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

2010-02-01

This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

Aromatase inhibitor regulates let-7 expression and let-7f induced cell migration in endometrial cells from women with endometriosis

PubMed Central

Cho, SiHyun; Mutlu, Levent; Zhou, Yuping; Taylor, Hugh S.

2018-01-01

Objectives To evaluate associations between aromatase inhibitor (AI) treatment and let-7 family microRNA expression in endometriosis. Design In vitro study using Ishikawa cells and human endometrial stromal cells (HESC) obtained from patients with endometriosis Setting University research center. Patients Women undergoing laparoscopic surgery for endometriosis Interventions HESCs and Ishikawa cells treated with various letrozol concentrations and transfected with a mimic of let-7 subtypes of interest Main Outcome Measures microRNAs let7a-f and aromatase expression were evaluated. Migration potential after transfection with a let-7f mimic were analyzed. Results After letrozole treatment for 48 hours, all let-7 subtypes showed a trend toward increased expression in a dose dependent manner in Ishikawa cells, and significant differences were found in let-7b and let-7f between controls and the 20 μmol/L treated groups. Further, let-7f showed significant differences between control and 1.0 μmol/L treatment group, a typical therapeutic level, in HESCs. Transfection of a let-7f mimic decreased aromatase expression in both Ishikawa cells and HESC, and led to a significant decrease in number of migrating cells in both cell types. Conclusions AI treatment significantly increased expression of let-7f in Ishikawa cells and HESCs from patient with endometriosis; increased lef-7f expression effectively reduced the migration of endometrial cells. Modulation of miRNAs involved in the pathogenesis of endometriosis may have therapeutic potential for endometriosis. PMID:27320036

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

PubMed Central

Takahashi, Shota; Asada, Atsushi; Matsuo, Minako; Kishikawa, Kenta; Mizuno, Akira

2015-01-01

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 103 cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy. PMID:26649904

[Construction and characterization of liposomal magnetofection system in pig kidney cells].

PubMed

Chen, Wenjie; Cui, Haixin; Zhao, Xiang; Cui, Jinhui; Wang, Yan; Sun, Changjiao

2014-06-01

Magnetic nano gene vector is one of the non-viral gene vectors, modified by functional group to bind cationic transfect reagents. Coupling magnetofection with the universal lipofection we developed a novel somatic cell transfection method as the so-called liposomal magnetofection (LMF). This approach is potential to provide somatic cell cloning with stable genetic cell lines to cultivate transgenic animals. In order to construct such liposomal magnetic gene vectors complexes system, we used nano magnetic gene vector to combine with liposomal cationic transfect reagents by molecular self-assembly. This vectors system successfully carried exogenous gene and then transfected animal somatic cells. Here, we conducted atomic force microscopy (AFM), zeta potential-diameter analysis and other characterization experiments to investegate the size distribution and morphology of magnetic nanoparticles, the way of the vectors to load and concentrate DNA molecules. Our data reveal that, the LMF of Pig Kidney cells exhibited higher transfection efficiency comparing with the transfection mediated by the commercial lipofectamine2000. Moreover, LMF method overcomes the constraint of transient expression mediated by lipofection. Meanwhile, MTT assay showed low cytotoxicity of LMF. Hence, LMF is a feasible, low cytotoxic and effective method of cell transfection.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

NASA Astrophysics Data System (ADS)

Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

2011-11-01

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

Nanoparticles as conjugated delivery agents for therapeutic applications

NASA Astrophysics Data System (ADS)

Muroski, Megan Elizabeth

This dissertation explores the use of nanoparticles as conjugated delivery agents. Chapter 1 is a general introduction. Chapter 2 discusses the delivery by a nanoparticle platform provides a method to manipulate gene activation, by taking advantage of the high surface area of a nanoparticle and the ability to selectively couple a desired biological moiety to the NP surface. The nanoparticle based transfection approach functions by controlled release of gene regulatory elements from a 6 nm AuNP (gold nanoparticle) surface. The endosomal release of the regulatory elements from the nanoparticle surface results in endogenous protein knockdown simultaneously with exogenous protein expression for the first 48 h. The use of fluorescent proteins as the endogenous and exogenous signals for protein expression enables the efficiency of co-delivery of siRNA (small interfering RNA) for GFP (green fluorescent protein) knockdown and a dsRed-express linearized plasmid for induction to be optically analyzed in CRL-2794, a human kidney cell line expressing an unstable green fluorescent protein. Delivery of the bimodal nanoparticle in cationic liposomes results in 20% GFP knockdown within 24 h of delivery and continues exhibiting knockdown for up to 48 h for the bimodal agent. Simultaneous dsRed expression is observed to initiate within the same time frame with expression levels reaching 34% after 25 days although cells have divided approximately 20 times, implying daughter cell transfection has occurred. Fluorescence cell sorting results in a stable colony, as demonstrated by Western blot analysis. The simultaneous delivery of siRNA and linearized plasmid DNA on the surface of a single nanocrystal provides a unique method for definitive genetic control within a single cell and leads to a very efficient cell transfection protocol. In Chapter 3, we wanted to understand the NP complex within the cell, and to look at the dynamics of release utilizing nanometal surface energy transfer as a molecular beacon. The development of non-viral transfection approaches using gold nanoparticles (AuNP) as a gene carrier allows the implementation of advanced biophysical tools to follow the transfection cycle by utilizing nanometal surface energy transfer (NSET) molecular beacon methods coupled to delivery of a gene that induces a fluorescent protein. The change in photoluminescence of an appended dye following gene release from the AuNP surface within endosomes can be tempo-rally and spatially followed. The ability to correlate the release events with the protein expression event by simultaneously monitoring fluorescent protein production provides insight into package uptake, nanoparticle disassembly, and final gene expression. Employing AuNP transfection constructs and then monitoring the stages of the transfection cycle via NSET, indicates delivery of the constructs leads to gene release from the AuNP surface within the endosome followed by slow cytosolic diffusion. The slow diffusion is the limiting step for transfection and impacts the protein yield due to competing degradation processes. Chapter 4 aims to improve the NP conjugate through the use of cell penetrating peptides (CPP) to Transfect Primary Cells. All future clinical applications of mesenchymal stem cell (MSC) therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to be able to systematically alter the protein expression in harvested MSC cells with high fidelity in a single transfection event. We have developed a bimodal delivery platform based on the use of a solid gold core nanoparticle that has been surface modified to produce a chimera containing a protein transduction domain (PTD) sequence to enhance cellular uptake and a linearized expression vector to induce protein production. The transfection chimera is observed to be an efficient inducer of protein expression following a single treatment of femur bone marrow isolated rat MSCs. Use of the neutral penta-peptide, Ku70, designed from Bax-inhibiting peptides in a 500:1 ratio to the linearized gene yields >80% transfection efficiencies. Chapter 5 further develops this idea by using cell penetrating peptides. Research over the past decade has identified several of the key limiting features in multidrug resistance therapy applications, such as, cellular targeting, protection from multidrug resistant mediators and retention of intact and functional drugs. Cell penetrating peptides are able to overcome the difficulties of drug transport resulting in improved efficacy of delivery. Functionalizing the cell penetrating peptide onto the surface of a quantum dot, allows the capability of creating an individualized package for further downstream studies. Four distinct cell penetrating peptides, TAT, VP-22, Ku-70, and hCT (9-32), were utilized to study the different profiles in gliosarcoma lines (rat 9L) with varying resistances to one of the most prescribed drugs in treating glioblastoma in the clinic; BCNU. (Abstract shortened by UMI.)

Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

PubMed

Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

2003-07-15

Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

[The concept of cellular immortality, a myth or a reality. Example of "immortalized" articular chondrocytes].

PubMed

Adolphe, M; Thenet, S

1990-01-01

The concept of cellular immortality, which arose from the historical studies of A. Carrel, is getting a new start with the progress of virology. However, the definition of cell immortalization is still ambiguous. Although scientists agree that cells regarded as immortal have acquired an infinite growth capacity, the relationship of this change with the first stages of transformation is difficult to clearly define. Immortalized cell lines have already been obtained from numerous cell types by using viral infection or transfection with viral and cellular genes. Immortalization of cells is interesting for three main reasons: it permits study of the steps in progression to transformation, allows establishment of cell lines for producing biological products, and permits various cell types to maintain a part of their differentiated functions. For example, hypothalamic neurosecretory cells, macrophages, astrocytes and intestinal epithelial cells have been immortalized and these lines can be used for understanding the balance between division and differentiation, and also for pharmacotoxicological studies. In our laboratory, we immortalized rabbit articular chondrocytes by transfection with SV40 large T and little t encoding genes. At the 9th subculture, when the control culture was senescent, clones of polygonal cells appeared in the transfected cell cultures. Three clones have been selected and have been maintained in culture for two years. Growth curves of normal and SV40-transfected chondrocytes were compared and displayed similar doubling times (approximately 20 hours). The exponential phase of growth was longer for immortalized cells resulting in a 2-fold higher saturation density. These cells appear to be not fully transformed and maintain some properties of differentiated chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation.

PubMed

Lo, Chia-Wen; Desjouy, Cyril; Chen, Shing-Ru; Lee, Jyun-Lin; Inserra, Claude; Béra, Jean-Christophe; Chen, Wen-Shiang

2014-03-01

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection. Copyright © 2013 Elsevier B.V. All rights reserved.

High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

NASA Astrophysics Data System (ADS)

Miller, Cynthia K.; Temin, Howard M.

1983-05-01

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Transcription of a vaccinia virus late promoter template: requirement for the product of the A2L intermediate-stage gene.

PubMed Central

Passarelli, A L; Kovacs, G R; Moss, B

1996-01-01

Evidence is presented that a 26-kDa protein encoded by the vaccinia virus A2L open reading frame, originally shown to be one of three intermediate-stage genes that together can transactivate late-stage gene expression in transfection assays (J. G. Keck, C. J. Baldick, and B. Moss, Cell 61:801-809, 1990), is required for in vitro transcription of a template with a late promoter. The critical step in this analysis was the preparation of an extract containing all the required factors except for the A2L protein. This extract was prepared from cells infected with a recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase in the presence of the DNA synthesis inhibitor cytosine arabinoside and transfected with plasmids containing the two other known transactivator genes, A1L and G8R, under T7 promoter control. Reaction mixtures made with extracts of these cells had background levels of late transcription activity, unless they were supplemented with extracts of cells transfected with the A2L gene. Active transcription mixtures were also made by mixing extracts from three sets of cells, each transfected with a gene (A1L, A2L, or G8R) encoding a separate factor, indicating the absence of any requirement for their coexpression. To minimize the possibility that the A2L protein functions indirectly by activating another viral or cellular protein, this gene was expressed in insect cells by using a baculovirus vector. The partially purified recombinant protein complemented the activity of A2L-deficient cell extracts. Recombinant A1L, A2L, and G8R proteins, all produced in insect cells, together complemented extracts from mammalian cells containing only viral early proteins, concordant with previous in vivo transfection data. PMID:8676468

Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism

PubMed Central

Harvey, Adam; Yen, Ten-Yang; Aizman, Irina; Tate, Ciara; Case, Casey

2013-01-01

Mesenchymal stromal cells (MSCs) transiently transfected with notch1 intracellular domain (NICD) are beneficial for neurological disorders as observed in several preclinical studies. Extracellular matrix (ECM) derived from NICD-transfected MSCs has been previously shown to support in vitro neural cell growth and survival better than that of un-transfected MSCs. To understand the underlying mechanism(s) by which NICD-transfected MSC-derived ECM supports neural cell growth and survival, we investigated the differences in NICD-transfected MSC- and MSC-derived ECM protein quantity and composition. To compare the ECM derived from MSCs and NICD-transfected MSCs, the proteins were sequentially solubilized using sodium dodecyl sulfate (SDS) and urea, quantified, and compared across four human donors. We then analyzed ECM proteins using either in-gel digests or in-solution surfactant-assisted trypsin digests (SAISD) coupled with reverse phase nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Analyses using nLC-MS/MS identified key components of ECM from NICD-transfected MSCs and un-transfected MSCs and revealed significant differences in their respective compositions. This work provides a reproducible method for identifying and comparing in vitro cell-derived ECM proteins, which is crucial for exploring the mechanisms underlying cellular therapy. PMID:24244468

PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

PubMed

Xu, Xiaolong; Qiu, Sujun; Zhang, Yuxian; Yin, Jie; Min, Shaoxiong

2017-03-01

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

Cotransfecting norepinephrine transporter and vesicular monoamine transporter 2 genes for increased retention of metaiodobenzylguanidine labeled with iodine 131 in malignant hepatocarcinoma cells.

PubMed

Zhao, Yanlin; Zhong, Xiao; Ou, Xiaohong; Cai, Huawei; Wu, Xiaoai; Huang, Rui

2017-03-01

Norepinephrine transporter (NET) transfection leads to significant uptake of iodine-131-labeled metaiodobenzylguanidine ( 131 I-MIBG) in non-neuroendocrine tumors. However, the use of 131 I-MIBG is limited by its short retention time in target cells. To prolong the retention of 131 I-MIBG in target cells, we infected hepatocarcinoma (HepG2) cells with Lentivirus-encoding human NET and vesicular monoamine transporter 2 (VMAT2) genes to obtain NET-expressing, NET-VMAT2-coexpressing, and negative-control cell lines. We evaluated the uptake and efflux of 131 I-MIBG both in vitro and in vivo in mice bearing transfected tumors. NET-expressing and NET-VMAT2-coexpressing cells respectively showed 2.24 and 2.22 times higher 131 I-MIBG uptake than controls. Two hours after removal of 131 I-MIBG-containing medium, 25.4% efflux was observed in NET-VMAT2-coexpressing cells and 38.6% in NET-expressing cells. In vivo experiments were performed in nude mice bearing transfected tumors; results revealed that NET-VMAT2-coexpressing tumors had longer 131 I-MIBG retention time than NET-expressing tumors. Meanwhile, NET-VMAT2-coexpressing and NET-expressing tumors displayed 0.54% and 0.19%, respectively, of the injected dose per gram of tissue 24 h after 131 I-MIBG administration. Cotransfection of HepG2 cells with NET and VMAT2 resulted in increased 131 I-MIBG uptake and retention. However, the degree of increase was insufficient to be therapeutically effective in target cells.

miR-181a promotes G1/S transition and cell proliferation in pediatric acute myeloid leukemia by targeting ATM.

PubMed

Liu, Xiaodan; Liao, Wang; Peng, Hongxia; Luo, Xuequn; Luo, Ziyan; Jiang, Hua; Xu, Ling

2016-01-01

Abnormal expression of miRNAs is intimately related to a variety of human cancers. The purpose of this study is to confirm the expression of miR-181a and elucidate its physiological function and mechanism in pediatric acute myeloid leukemia (AML). Pediatric AML patients and healthy controls were enrolled, and the expression of miR-181a and ataxia telangiectasia mutated (ATM) in tissues were examined using quantitative PCR. Moreover, cell proliferation and cell cycle were evaluated in several cell lines (HL60, NB4 and K562) by using flow cytometry after transfected with miR-181a mimics and inhibitors, or ATM siRNA and control siRNA. Finally, ATM as the potential target protein of miR-181a was examined. We found that miR-181a was significantly increased in pediatric AML, which showed an inverse association with ATM expression. Overexpressed miR-181a in cell lines significantly enhanced cell proliferation, as well as increased the ratio of S-phase cells by miR-181a mimics transfection in vitro. Luciferase activity of the reporter construct identified ATM as the direct molecular target of miR-181a. ATM siRNA transfection significantly enhanced cell proliferation and increased the ratio of S-phase cells in vitro. The results revealed novel mechanism through which miR-181a regulates G1/S transition and cell proliferation in pediatric AML by regulating the tumor suppressor ATM, providing insights into the molecular mechanism in pediatric AML.

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

PubMed Central

Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies. PMID:26407291

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.

PubMed

Ker, Dai Fei Elmer; Sharma, Rashmi; Wang, Evelyna Tsi Hsin; Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

B29 Gene Silencing in Pituitary Cells is Regulated by Its 3′ Enhancer

PubMed Central

Malone, Cindy S.; Kuraishy, Ali I.; Fike, Francesca M.; Loya, Ruchika G.; Mikkili, Minil R.; Teitell, Michael A.; Wall, Randolph

2007-01-01

Summary B cell-specific B29 (Igβ, CD79b) genes in rat, mouse, and human are situated between the 5′ growth hormone (GH) locus control region (LCR) and the 3′ GH gene cluster. The entire GH genomic region is DNase1 hypersensitive in GH-expressing pituitary cells, which predicts an “open” chromatin configuration, and yet B29 is not expressed. The B29 promoter and enhancers exhibit histone deacetylation in pituitary cells, but histone deacetylase inhibition failed to activate B29 expression. The B29 promoter and a 3′ enhancer showed local dense DNA methylation in both pituitary and non-lymphoid cells consistent with gene silencing. However, DNA methyltransferase inhibition did not activate B29 expression either. B29 promoter constructs were minimally activated in transfected pituitary cells. Co-transfection of the B cell-specific octamer transcriptional co-activator Bob1 with the B29 promoter construct resulted in high level promoter activity in pituitary cells comparable to B29 promoter activity in transfected B cells. Unexpectedly, inclusion of the B29 3′ enhancer in B29 promoter constructs strongly inhibited B29 transcriptional activity even when pituitary cells were co-transfected with Bob1. Both Oct-1 and Pit-1 bind the B29 3′ enhancer in in vitro EMSA and in in vivo chromatin immunoprecipitation analyses. These data indicate that the GH locus-embedded, tissue-specific B29 gene is silenced in GH-expressing pituitary cells by epigenetic mechanisms, the lack of a B cell-specific transcription factor, and likely by the B29 3′ enhancer acting as a powerful silencer in a context and tissue-specific manner. PMID:16920149

Molecular cloning and preliminary function study of iron responsive element binding protein 1 gene from cypermethrin-resistant Culex pipiens pallens

PubMed Central

2011-01-01

Background Insecticide resistance jeopardizes the control of mosquito populations and mosquito-borne disease control, which creates a major public health concern. Two-dimensional electrophoresis identified one protein segment with high sequence homology to part of Aedes aegypti iron-responsive element binding protein (IRE-BP). Method RT-PCR and RACE (rapid amplification of cDNA end) were used to clone a cDNA encoding full length IRE-BP 1. Real-time quantitative RT-PCR was used to evaluate the transcriptional level changes in the Cr-IRE strain Aedes aegypti compared to the susceptible strain of Cx. pipiens pallens. The expression profile of the gene was established in the mosquito life cycle. Methyl tritiated thymidine (3H-TdR) was used to observe the cypermethrin resistance changes in C6/36 cells containing the stably transfected IRE-BP 1 gene of Cx. pipiens pallens. Results The complete sequence of iron responsive element binding protein 1 (IRE-BP 1) has been cloned from the cypermethrin-resistant strain of Culex pipiens pallens (Cr-IRE strain). Quantitative RT-PCR analysis indicated that the IRE-BP 1 transcription level was 6.7 times higher in the Cr-IRE strain than in the susceptible strain of 4th instar larvae. The IRE-BP 1 expression was also found to be consistently higher throughout the life cycle of the Cr-IRE strain. A protein of predicted size 109.4 kDa has been detected by Western blotting in IRE-BP 1-transfected mosquito C6/36 cells. These IRE-BP 1-transfected cells also showed enhanced cypermethrin resistance compared to null-transfected or plasmid vector-transfected cells as determined by 3H-TdR incorporation. Conclusion IRE-BP 1 is expressed at higher levels in the Cr-IRE strain, and may confer some insecticide resistance in Cx. pipiens pallens. PMID:22075242

Syndecan-1 alterations during the tumorigenic progression of human colonic Caco-2 cells induced by human Ha-ras or polyoma middle T oncogenes.

PubMed Central

Levy, P.; Munier, A.; Baron-Delage, S.; Di Gioia, Y.; Gespach, C.; Capeau, J.; Cherqui, G.

1996-01-01

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras and pp60c-src in human colonic Caco-2 cells is associated with specific alterations of syndecan-1, a membrane-anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated (Val 12) human Ha-ras gene or with the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines (1) contained smaller amounts of membrane-anchored PGs; (2) exhibited decreased syndecan-1 expression at the protein but not the mRNA level; (3) synthesized 35S-labelled syndecan-1 with decreased specific activity; (4) produced a syndecan-1 ectodomain with a lower molecular mass and reduced GAG chain size and sulphation; and (5) expressed heparanase degradative activity. These results show that the dramatic activation of the tumorigenic potential induced by oncogenic p21ras or Py-MT/pp60c-src in Caco-2 cells is associated with marked alterations of syndecan-1 expression at the translational and post-translational levels. Images Figure 2 PMID:8695359

Enhancement and optimization of plasmid expression in femtosecond optical transfection.

PubMed

Praveen, Bavishna B; Stevenson, David J; Antkowiak, Maciej; Dholakia, Kishan; Gunn-Moore, Frank J

2011-04-01

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non-invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3-fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO-K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ~70% without compromising the transfection efficiency. Also, we report for the first time the successful photo-transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

PubMed

Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

2012-12-15

The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

Temporal Control of Transforming Growth Factor (TGF) - Betal Expression on Mammary Cell Multistep Transformation

DTIC Science & Technology

1999-10-01

deregulated cell mors and may eventually regress through growth (18). The importance of APC and 0- cat - apoptosis (25). enin in the development of colorectal...progression. In support of this idea, Torre Amione et al. [74] demonstrated that, unlike parental tumor cells, fibrosarcoma cells transfected to express 10

African swine fever virus IAP-like protein induces the activation of nuclear factor kappa B.

PubMed

Rodríguez, Clara I; Nogal, María L; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2002-04-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

PubMed Central

Rodríguez, Clara I.; Nogal, María L.; Carrascosa, Angel L.; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

2002-01-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2. PMID:11907233

Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication.

PubMed

Konopka, K; Lee, N S; Rossi, J; Düzgüneş, N

2000-09-19

We examined whether the antiviral effect of an HIV-1 Rev-binding aptamer [RBE(apt)] could be enhanced by a ribozyme directed against the HIV-1 env gene, and whether the antiviral activity was affected by different promoters. The efficacy of the aptamer and ribozyme DNAs was tested in HeLa cells co-transfected with the HIV-1 proviral clones, HXBDeltaBgl or pNL4-3, using transferrin-lipoplexes. The RBE(apt) and anti-env ribozyme genes were inserted into the pTZU6+27 plasmid, or constructed under the control of the human cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. The parental vector plasmids were used as controls. Co-transfection of the pTZU6+27 RBE(apt) plasmid with HXBDeltaBgl, or pNL4-3, at a weight ratio of 5:1, inhibited p24 production by 70 and 45%, respectively. The RSV RBE(apt) plasmid co-transfected with either HIV clone, at the same weight ratio, reduced viral production by 88%. The addition of the anti-env ribozyme to the RSV RBE(apt) did not enhance its antiviral activity. When the constructs were under the control of the CMV promoter, the expression of the HIV plasmids was very low and was independent of the presence of the RBE(apt). Thus, the effect of the RBE(apt) was strongly dependent on the promoter of the tested construct. The anti-HIV activity of the CMV RBE(apt) construct was non-specific, because co-transfection with either pCMV. SPORT-betagal or pCMVlacZ significantly suppressed HIV production from the HIV proviral clones. The reduction in p24 could not be attributed to the non-specific toxicity of the transfection procedure. Transfection of acutely HIV-infected HeLa-CD4 cells with pCMV.SPORT-betagal reduced the p24 level by 35%, while the expression of the U6 RBE(apt) did not affect p24 production. The suppression of HIV production from the HIV proviral clones by the CMV promoter constructs in the co-transfection assays may be explained by competition for transcription factors (TFs) between HIV and CMV promoters. This observation points to the potential for misleading results in co-transfections involving CMV constructs and HIV.

Anti-miR21 oligonucleotide enhances chemosensitivity of T98G cell line to doxorubicin by inducing apoptosis

PubMed Central

Giunti, Laura; da Ros, Martina; Vinci, Serena; Gelmini, Stefania; Iorio, Anna Lisa; Buccoliero, Anna Maria; Cardellicchio, Stefania; Castiglione, Francesca; Genitori, Lorenzo; de Martino, Maurizio; Giglio, Sabrina; Genuardi, Maurizio; Sardi, Iacopo

2015-01-01

Various signal transduction pathways seem to be involved in chemoresistance mechanism of glioblastomas (GBMs). miR-21 is an important oncogenic miRNA which modulates drug resistance of tumor cells. We analyzed the expression of 5 miRNAs, previously found to be dysregulated in high grade gliomas, in 9 pediatric (pGBM) and in 5 adult (aGBM) GBMs. miR-21 was over-expressed, with a significant difference between pGBMs and aGBMs represented by a 4 times lower degree of expression in the pediatric compared to the adult series (p = 0.001). Doxorubicin (Dox) seems to be an effective anti-glioma agent with high antitumor activity also against glioblastoma stem cells. We therefore evaluated the chemosensitivity to Dox in 3 GBM cell lines (A172, U87MG and T98G). Dox had a cytotoxic effect after 48 h of treatment in A172 and U87MG, while T98G cells were resistant. TUNEL assay verified that Dox induced apoptosis in A172 and U87MG but not in T98G. miR-21 showed a low basal expression in treated cells and was over-expressed in untreated cells. To validate the possible association of miR-21 with drug resistance of T98G cells, we transfected anti-miR-21 inhibitor into the cells. The expression level of miR-21 was significantly lower in T98G transfected cells (than in the parental control cells). Transfected cells showed a high apoptotic rate compared to control after Dox treatment by TUNEL assay, suggesting that combined Dox and miR-21 inhibitor therapy can sensitize GBM resistant cells to anthracyclines by enhancing apoptosis. PMID:25628933

Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line

PubMed Central

Song, Xiu-Guang; Bian, Peng-Fei; Yu, Shu-Li; Zhao, Xiu-Hua; Xu, Wei; Bu, Xue-Hui; Li, Xia; Ma, Li-Xian

2013-01-01

AIM: To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. METHODS: Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. RESULTS: The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h after transfection. The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells. HBV DNA replication intermediates were also observed at 72 h after transfection, including relaxed circular DNA, double-stranded DNA and single-stranded DNA. Furthermore, a few 42 nm Dane particles, as well as many 22 nm subviral particles with a spherical or filamentous shape, were detected in the supernatant. CONCLUSION: SV40T expression can immortalize mouse hepatic cells, and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model. PMID:24307795

Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line.

PubMed

Song, Xiu-Guang; Bian, Peng-Fei; Yu, Shu-Li; Zhao, Xiu-Hua; Xu, Wei; Bu, Xue-Hui; Li, Xia; Ma, Li-Xian

2013-11-28

To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h after transfection. The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells. HBV DNA replication intermediates were also observed at 72 h after transfection, including relaxed circular DNA, double-stranded DNA and single-stranded DNA. Furthermore, a few 42 nm Dane particles, as well as many 22 nm subviral particles with a spherical or filamentous shape, were detected in the supernatant. SV40T expression can immortalize mouse hepatic cells, and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.

Software-aided automatic laser optoporation and transfection of cells

PubMed Central

Georg Breunig, Hans; Uchugonova, Aisada; Batista, Ana; König, Karsten

2015-01-01

Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates. PMID:26053047

Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

PubMed Central

McPhaul, M; Berg, P

1986-01-01

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes. Images PMID:3466162

Inhibition of the expression of aquaporin‑1 by RNA interference in pulmonary epithelial cells and its effects on water transport.

PubMed

Zhang, Qiuyue; Fu, Jianhua; Xue, Xindong

2016-01-01

In the present study, the effect of aquaporin‑1 (AQP1) on fluid transportation in pulmonary epithelial cells, and the role of AQP1 in alveolar fluid clearance were investigated to provide an experimental foundation to elucidate the pathogenesis of hyperoxic lung edema. An siRNA transfection technique was used to silence AQP1 in the A549 cell line. The transfected cells were randomized into a hyperoxia exposure and an air control group, with a negative control group set for each group. Cell volume was determined using flow cytometry, and Pf values were used to determine osmotic water permeability. Cell volume was found to be reduced in the AQP1‑silenced A549 cells, compared with the negative control group 72 h following air exposure. In addition, cell volume was reduced in the AQP1‑silenced A549 cells, compared with the negative control group 48 and 72 h following hyperoxia exposure. The osmotic water permeability of the AQP1‑silenced cells was reduced in the air control and hyperoxia exposure groups, compared with the negative control group 48 and 72 h following exposure. The volume and cell membrane osmotic water permeability of the A549 cells were reduced, compared with those in the control group following AQP1‑silencing, which indicated that the downregulation of AQP1 impedes extracellular to intracellular fluid transportation. Therefore, the disturbance in alveolar fluid clearance resulting from the downregulation of AQP1 following hyperoxia exposure may be one of the key mechanisms responsible for hyperoxic lung edema.

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

PubMed Central

2010-01-01

Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

PubMed

Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

2014-07-01

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

[Optimizing condition for oligofectamine-mediated SP1 decoy oligodeoxynucleotides transfection into SV-40-PED cells].

PubMed

Huang, Yabing; Xu, Jing; Chen, Song

2007-08-01

To determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. With a change of Decoy oligodeoxynucleotides (ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 microl oligofectamine with different concentrations of ODNs(2.5, 5.0, 7.5, 10.0 and 12.5 microl) were put into 100 microl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50,100, 150, 200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution of SP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity. The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a stronger fluorescence intensity with a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12+/-3.92 U/L in the 72-h group, which was significantly higher those that in the 26-h group (49.61+/-17.13 U/L) and the 48-h group (120.26 +/- 8.42 U/L) (P<0.01). At 26 h after the transfection, there were no statistical differences at the above LDH levels in the different-concentration groups (P > 0.05). Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.

miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy.

PubMed

Gao, Shiqian; Tian, Huayu; Guo, Ye; Li, Yuce; Guo, Zhaopei; Zhu, Xiaojuan; Chen, Xuesi

2015-10-01

MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer combines the high transfection efficiency of polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post transfection. Compared with the negative control, PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4 (PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. This work was a combination of the high transfection efficiency of polyethylenimine (PEI), the good biodegradability of polyllysine (PLL) and the breast cancer-killing effect of miR-21 inhibitors. The poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer was employed as the vector of miR-21 sponge plasmid DNA (Sponge) or anti-miR-21 oligonucleotide (AMO). PEI-PLL showed more transfection efficiency and lower cytotoxicity in human breast cancer cells than PEI. Moreover, the breast cancer cells exhibited significantly lower miR-21 expression and cell viability post transfection with sponge or AMO. Interestingly, the PEI-PLL/miR-21 inhibitor complexes also sensitized the cancer cells to anti-cancer chemotherapy drugs, doxorubicin (DOX) and cisplatin (CDDP). This synergistic effect provides a good application prospect of co-delivery miR-21 inhibitors and chemical drugs in breast cancer therapy. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

GARP is regulated by miRNAs and controls latent TGF-β1 production by human regulatory T cells.

PubMed

Gauthy, Emilie; Cuende, Julia; Stockis, Julie; Huygens, Caroline; Lethé, Bernard; Collet, Jean-François; Bommer, Guido; Coulie, Pierre G; Lucas, Sophie

2013-01-01

GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-β1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-β1 precursor and increases the amount of secreted latent TGF-β1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-β1 that is disulfide-linked to GARP. These GARP/TGF-β1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-β1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-β1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3' UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-β1, and secretion of latent TGF-β1.

[Effects of HOXB2 antisense oligodeoxynucleotides on the biological properties of primary human umbilical vein endothelial cells].

PubMed

Zhang, Xiaoqi; Liu, Xusheng

2002-07-01

To explore the effects of HOXB2 antisense oligodeoxynuc leotides (Asodn) on the biological properties of primary human umbilical vein endothelial cells (ECs). Fluorescent labelled Asodn was transfected into the endothelial cells of human unbilical vein mediated liposome and its distribution within endothelia was observed. (3)H-TdR incorporation test was employed to determine its effects on the DNA synthesis. Flow cytometry was applied to determine the change of the cell cycle. In the same time, RT-PCR was adopted to study the influence of Asodn on the expression of target genes. Fifteen minutes after the transfection, weak nucleic staining was observed. The fluorescent staining was the strongest 4 approximately 8 hours after the transfection and began to weaken in 16 hours. The proportion of cells in G1/0 phase in Asodn group was 53.4 +/- 3.1, significantly higher than that in control group (35.8 +/- 7.3, P < 0.01), and the proportion of cells in S phase in Asodn group was 42.2 +/- 3.5, significantly lower than that in control group (60.8 +/- 6.2, P < 0.01). The expression of HOXB2 mRNA was remarkably decreased during 24 to 48 hours. HOXB2 Asodn exerts inhibitory effects on EC proliferation dose-dependently, delays the conversion of G1 phase to S Phase, and inhibits the expression of HOXB2 mRNA. HOXB2 gene plays an important role in proliferation of endothelial cells and the mechanism is related to cell cycle.

A caspase 8-based suicide switch induces apoptosis in nanobody-directed chimeric receptor expressing T cells.

PubMed

Khaleghi, Sepideh; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J; Pognonec, Philippe

2012-04-01

In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-stimulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.

Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

PubMed

Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

2016-01-01

Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.

Sodium-iodine symporter gene expression controlled by the EGR-1 promoter: biodistribution, imaging and in vitro radionuclide therapy with Na(131)I.

PubMed

Tang, Jun; Wang, Xiaoxia; Xu, Yuanqi; Shi, Yizhen; Liu, Zengli; Yang, Yi

2015-02-01

The objective of this study is to explore the feasibility of radioiodine treatment for cervical cancer using the early growth response (Egr-1) promoter to control sodium-iodine symporter (hNIS) gene expression. The hNIS gene was previously transfected into Hela cells under the control of either the cytomegalovirus (CMV) or Egr-1 promoters. Na(125)I uptake was measured in the presence or absence of NaClO4. Na(125)I efflux was measured. The effects of external beam radiation on iodine uptake and retention were studied. The cytotoxic effects of (131)I were measured by clonogenic assay. The Na(125)I biodistribution was obtained using mice bearing control and transfected cells. The %ID/g of tumor and major organs were obtained for a range of times up to 48 hours post injection and the ratio of tumor to non-tumor activity (T/NT) was calculated. Tumors were imaged with Na(131)I and (99m)TcO4 (-), and the ratio of tumor to background activity (T/B) was calculated. Na(125)I uptake in Hela cells was minimal in the absence of hNIS. Uptake in the transfected cells was strong, and could be blocked by NaClO4. The iodine uptake of Hela-Egr-1-hNIS cells increased after the irradiation, and the magnitude of this effect approximately matched the radiation dose delivered. The efflux of 125I was affected by neither the promoter sequence nor pre-irradiation. (131)I reduced the clonogenic survival of symporter expressing cells, relative to the parental line. The effect was greatest in cells where hNIS was driven by the CMV promoter. Tumors formed from Hela-Egr-1-hNIS concentrated Na(125)I over a 12 hour period, in contrast to untransfected cells. These tumors could also be successfully imaged using either Na(131)I or (99m)TcO4 (-). (131)I uptake peaked at 4h, while (99m)TcO4 (-) accumulated over approximately 20 hours. In vivo uptake of (131)I and (99m)TcO4 (-) was slightly higher in cells transfected with the Egr-1 promoter, compared to CMV. Hela-Egr-1-hNIS cells demonstrate highly enhanced iodine uptake, and this effect is further augmented by radiation, creating a positive feedback loop which may bolster radionuclide therapy in vivo. © The Author(s) 2014.

SLC4A11 is an EIPA-sensitive Na+ permeable pHi regulator

PubMed Central

Ogando, Diego G.; Jalimarada, Supriya S.; Zhang, Wenlin; Vithana, Eranga N.

2013-01-01

Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na+-B(OH)4− (borate) cotransporter and a Na+-2OH− cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na+-HCO3− cotransporter, 2) Na+-OH−(H+) transporter, and/or 3) Na+-B(OH)4− cotransporter. CO2/HCO3− perfusion yielded no significant differences in rate or extent of pHi changes or Na+ flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3−, acidification on removal of Na+ and alkalinization on Na+ add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na+-HCO3− cotransport activity. In the absence of CO2/HCO3−, SLC4A11-transfected cells showed higher resting intracelllular Na+ concentration ([Na+]i; 25 vs. 17 mM), increased NH4+-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na+ efflux and influx were faster (80%) following Na+ removal and add back, respectively, indicative of Na+-OH−(H+) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na+-OH−(H+) transporter and not an activator of NHEs. SLC4A11-mediated H+ efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 μM). The presence of 10 mM borate did not alter dpHi/dt or ΔpH during a Na+-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na+-OH−(H+) and NH4+ permeability. PMID:23864606

[Experimental study on dog's bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene].

PubMed

Zhu, Guo-qiang; Wu, Zhi-fen; Li, Yuan-fei; Hu, De-hua; Wang, Qin-tao

2006-12-01

To establish the bone marrow stem cells (MSC) model which could highly express the insulin-like growth factor 1 (IGF-1) transfected by dog's IGF-1 gene. pIRES2-EGFP-IGF-1 was transfected into MSC by lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSC was detected by sandwich-in ELISA. The periodontal ligament cells (PDLC) were cultured in the supernatant of the transfected MSC. The changes of PDLC' proliferation were observed by MTT. IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC' proliferation. The MSC transfected by dog's IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.

[The influence of EphA2 overexpression on proliferation and apoptosis of human lens epithelial cells exposed to high-concentration dexamethasone in vitro].

PubMed

Ma, C X; Zheng, G Y

2018-02-11

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. The 293 T/17 cells were co-transfected with the pCDH-CMV-MCS-EF1-RFP-EphA2 and the packaging mixture by Lipofectamine 2000. At different multiplicities of infection (MOI=20, 50, 100, and 200) after 72-hour infection, we observed the expression of RFP and morphological changes of HLE-B3 by an inverted fluorescence microscope, and calculated the transfection efficiency through the flow cytometry. EphA2 protein expression was detected by Western blot. The following experiments were divided into four groups: normal control group (group A), EphA2 overexpression vector transfection group (group B), HLE-B3 cells exposed to dexamethasone group (group C) and EphA2 overexpression vector transfection HLE-B3 cells exposed to dexamethasone group (group D). Statistical analysis method was single factor or two factors variance analysis. Cell survival rate was detected by the Cell Counting Kit-8 assay. Cell apoptosis index was detected by Tunel. Results: Restriction enzyme digestion and sequencing indicated that EphA2 cDNA fragment was successfully inserted in the vector. The infection efficiency was up to 38.6%±3.9%, 49.2%±4.2%, 79.5%±5.5% and 80.2%±6.0% when the MOI was 20, 50, 100 and 200, respectively. There was statistically significant difference ( F= 2 600.8, P= 0.001) among the four groups and between any two groups except between the MOI=100 group and MOI=200 group ( P= 2.507) . The relative quantity of EphA2 protein of the normal control group, empty vector transfection group and EphA2 gene overexpression vector transfection group was (0.561 2±0.031 7) , (0.559 7±0.012 8) and (3.032 0±0.041 9) , respectively. There was statistically significant difference ( F= 2 646.0, P= 0.001) among the three groups and between any two groups except between the normal control group and empty vector transfection group ( P= 0.868) . The survival rate of groups A, B, C and D was 98.18%±1.85%, 122.01%±3.89%, 52.32%±1.99% and 76.18%±3.74%, respectively. There was statistically significant difference among the four groups ( F= 497.6, P= 0.001) . The survival rate of group B was greater than group A ( P= 0.001) . The survival rate of group D was greater than group C ( P= 0.001) . Tunel results showed that the apoptosis index of groups A, B, C and D was 5.4%±1.5%, 5.0%±1.3%, 23.0%±3.9% and 14.4%±2.7%, respectively. There was statistically significant difference among the four groups ( F= 397.6, P= 0.001) . The apoptosis index of group B was lower than group A, but there was no statistically significant difference between them ( P= 0.415) ; the apoptosis index of group D was lower than group C ( P= 0.018). Conclusions: The lentiviral vector carrying human EphA2 gene has been successfully constructed and efficiently expressed in HLE-B3 cells. EphA2 gene overexpression could increase the HLE-B3 cell survival rate and protect HLE-B3 cells from high-concentration dexamethasone-induced reduction of the cell survival rate. EphA2 gene overexpression could protect HLE-B3 cells from high-concentration dexamethasone-induced apoptosis, but it has no remarkable effect on apoptosis of HLE-B3 cells under physiological conditions. (Chin J Ophthalmol, 2018, 54: 125-132) .

[EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS].

PubMed

Yu, Fengxu; Chen, Yongen; Chen, Feng; Xia, Jiyi; Liu, Hongduan; Fu, Yong; Li, Miaoling; Liao, Bin

2016-04-01

To investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. LVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3PLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94% N₂, 1% O₂, and 5% CO₂ for 0, 3, 6, 9, and 12 hours (group B1 and group C1). The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t = 17.525, P = 0.013). The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P < 0.05). In group C1, the apoptosis rate and cell death rate decreased temporarily at 3 hours after hypoxia intervention, then increased gradually, and difference was significant (P < 0.05). The apoptosis rate and cell death rate of group C1 were significantly lower than those of group B1 at each time point (P < 0.05) except at 0 hour. MTT assay showed tat absorbance (A) values of groups B and C were significantly higher than those of groups B1 and C1 at each time point (P < 0.05); the A value of group B was significantly lower than that of group C at each time point (P < 0.05). The A value of group B1 was significantly lower than that of group Cl at 6, 9, and 12 hours after hypoxia intervention (P < 0.05). Western blot results showed that the Caspase-3 expression of group C1 significantly reduced when compared with group B1 at each time point (P < 0.05). Akt1 gene transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

PubMed

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Smart Carbon Nanotubes with Laser-Controlled Behavior in Gene Delivery and Therapy through a Non-Digestive Trafficking Pathway.

PubMed

Kong, Fenfen; Liu, Fei; Li, Wei; Guo, Xiaomeng; Wang, Zuhua; Zhang, Hanbo; Li, Qingpo; Luo, Lihua; Du, Yongzhong; Jin, Yi; You, Jian

2016-12-01

Near-infrared (NIR) laser-controlled gene delivery presents some benefits in gene therapy, inducing enhanced gene transfection efficiency. In this study, a "photothermal transfection" agent is obtained by wrapping poly(ethylenimine)-cholesterol derivatives (PEI-Chol) around single-walled carbon nanotubes (SWNTs). The PEI-Chol modified SWNTs (PCS) are effective in compressing DNA molecules and protecting them from DNaseI degradation. Compared to the complexes formed by PEI with DNA (PEI/DNA), complexes of PCS and DNA that are formed (PCS/DNA) exhibit a little lower toxicity to HEK293 and HeLa cells under the same PEI molecule weight and weight ratios. Notably, caveolae-mediated cellular uptake of PCS/DNA occurs, which results in a safer intracellular transport of the gene due to the decreased lysosomal degradation in comparison with that of PEI/DNA whose internalization mainly depends on clathrin rather than caveolae. Furthermore, unlike PEI/DNA, PCS/DNA exhibits a photothermal conversion ability, which promotes DNA release from PCS under NIR laser irradiation. The NIR laser-mediated photothermal transfection of PCS 10K /plasmid TP53 (pTP53) results in more apoptosis and necrosis of HeLa cells in vitro than other groups, and achieves a higher tumor-growth inhibition in vivo than naked pTP53, PEI 25K /pTP53, and PCS 10K /pTP53 alone. The enhanced transfection efficiency of PCS/DNA can be attributed to more efficient DNA internalization into the tumor cells, promotes detachment of DNA from PCS under the mediation of NIR laser and higher DNA stability in the cells due to caveolae-mediated cellular uptake of the complexes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Downregulation of Pygopus 2 inhibits vascular mimicry in glioma U251 cells by suppressing the canonical Wnt signaling pathway

PubMed Central

WANG, HAIDONG; FU, JIANHUA; XU, DIANSHUANG; XU, WEIWEI; WANG, SHIYONG; ZHANG, LIU; XIANG, YONGSHENG

2016-01-01

Gliomas are the most common type of malignant primary brain tumor, and the Wnt signaling pathway is associated with glioma malignancy. Pygopus protein plays an important role in developmental brain patterning, and has been identified to be a component of the Wnt signaling pathway. In the present study, the Pygopus 2 (Pygo2) protein was examined in 80 glioma tissue samples. Short hairpin (sh)RNA-Pygo2 was transfected into glioma U251 cells, and the cell proliferation, colony formation and bromodeoxyuridine (BrdU) incorporation were analyzed. Western blot analysis and reverse transcription-polymerase chain reaction were used to detect the expression of Pygo2. A vascular mimicry assay was performed to examine the vascular mimicry of U251 cells. A luciferase reporter assay was used to detect the β-catenin/Wnt system. The cyclin D1 protein was also detected using western blot analysis. The results demonstrated that inhibition of the expression of Pygo2 significantly triggered the decrease of cell proliferation, colony formation and BrdU incorporation compared with the cells treated with scramble control shRNA (shRNA-Scr). shRNA-Pygo2 transfection was found to inhibit vascular-mimicry and block the Wnt signaling pathway compared to the cells transfected with shRNA-Scr. The transfection of shRNA-Pygo2 also decreased the expression of the Wnt target gene cyclin D1. In conclusion, shRNA-Pygo2 suppressed glioma cell proliferation effectively and inhibited vascular mimicry by inhibiting the expression of cyclin D1 in the canonical Wnt/β-catenin pathway in brain glioma cells. PMID:26870266

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

PubMed

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

PubMed

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

[Effects of different nuclear factor kappaB dimers on the survival of immortalized neural progenitor cells].

PubMed

Gui, Ling-Li; Zhang, Chuan-Han; Liu, Zhi-Heng; Chen, Zhao-Jun; Zhu, Chang

2008-04-01

To investigate the effects of different nuclear factor (NF)-KB dimers on the survival of immortalized neural progenitor cells (INPCs). The control vector RC/CMV, containing the promoter of cytomegalovirus (CMV), and the expression vectors, RcCMV-p50 and RcCMV-p65, containing the coding regions of NF-KB subunits p50 and p65 genes, were transfected into the INPCs by liposome respectively. Stably transfected clones were screened out following G418 selection. Subsequently, the plasmid RcCMV-p50 was transiently transfected into the INPCs which had been stably transfected with the plasmid RcCMV-p65. The expression of p50 or p65 gene was detected in each cell strain by Western blotting. And the NF-KB DNA binding activity in the cell nuclear extracts was measured by electrophoresis mobility shift assay (EMSA). The expression of IkappaBalpha in the cytoplasm was detected by Western blotting. After oxygen and glucose deprivation for 13 h, the cell survival rate was measured by MTT assay. After gene transfection, five different cell strains were obtained: INPC, INPC/CMV, INPC/p50, INPC/p65, and INPC/p50p65. p50 or p65 gene was translated correctly and efficiently in the cell strains which had been transfected with the corresponding plasmids. EMSA showed that the INPC/p50, INPC/p65, and INPC/p50p65 cells all gave rise to NF-kappaB specific bands, which were composed of p50 homodimer, p65 homodimer, and p50 p65 heterodimer and p50 homodimer respectively. The expression of IkappaBbeta was increased significantly in the cytoplasm of the INPC/p65 and INPC/p50p65 cells. Games-Howell test showed that after oxygen and glucose deprivation for 13 h, the survival rates of the NPC/p65 and INPC/p50p65 cells were (6.0 +/- 1.0)% and (4.6 +/- 0.6)% respectively, both significantly lower than those of the INPC, INPC/CMV, and INPC/p50 cells [(72.5 +/- 6.2)%, (70.1 +/- 4.3)%, and (70.4 +/- 7.3)% respectively, all P < 0.05]. Overexpression of p50 gene and p65 gene directly enhance the DNA binding activities of different NF-kappaB dimers in the nuclei. In neural progenitor cells, NF-kappaB dimers with transcriptive activity decreases the cellular survival after oxygen and glucose deprivation, but NF-kappaB dimers without transcriptive activity don't prevent the cells from death.

Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO2 technique.

PubMed

Heyde, Mieke; Partridge, Kris A; Howdle, Steven M; Oreffo, Richard O C; Garnett, Martin C; Shakesheff, Kevin M

2007-10-15

Polyamidoamine polymers (PAA) comprising methylene-bisacrylamide/dimethylethylene-diamine monomers were synthesized, complexed with DNA and incorporated into porous P(DL)LA scaffolds by using a supercritical CO(2) (scCO(2)) technique. Scaffolds were made in a dry state consequently there was a need to lyophilize the complexes. A statistically significant reduction of the transfection efficiency was observed in the absence of trehalose when compared to the original complex after freeze-drying. Increasing concentrations (0-10% w/v) of trehalose were added to the complex prior to freeze-drying. Structure dependent differences in DNA binding were evaluated by gel electrophoresis and thermal transition analysis. TEM and PCS showed aggregate formation after freeze-drying without trehalose. Scaffolds were characterized by pore sizes of 173 +/- 73 microm and a porosity of 71%. The transfection potential of the released DNA was investigated by seeding scaffolds with A549 cells and following firefly luciferase as a marker gene after 48 h exposure. Low but continuous levels of transfection were observed for PAA complexes during a 60-day study. Complexes made with Lipofectaminetrade mark gave initially higher levels of DNA release but no further expression was seen after 40 days. Uncomplexed DNA showed background levels of transfection. Culturing cells on 3D scaffolds showed a benefit in retention of transfection activity with time compared to 2D controls. Transfection levels could be increased when cells were grown in OptiMEM. This study demonstrated that PAA/DNA complexes incorporated into a P(DL)LA scaffold made by using scCO(2) processing exhibited a slow release and extended gene expression profile. Copyright 2007 Wiley Periodicals, Inc.

Human Mammary Epithelial Cell Transformation by Rho GTPase Through a Novel Mechanism

DTIC Science & Technology

2009-08-01

cells were transfected with myc-tagged Prks -ROCK1 or Prks -ROCK2, flag-tagged Prc-PKN-AL, or mDia1 using calcium phosphate method. The transfectants...plasmids, Prks -ROCK1, Prks -ROCK2, Prc-PKN-AL, and pFL-mDia1, were transfected into 293T cells, and cell lysates were incubated with GTP-g-S–loaded GST

Shock Wave-Induced Damage and Poration in Eukaryotic Cell Membranes.

PubMed

López-Marín, Luz M; Millán-Chiu, Blanca E; Castaño-González, Karen; Aceves, Carmen; Fernández, Francisco; Varela-Echavarría, Alfredo; Loske, Achim M

2017-02-01

Shock waves are known to permeabilize eukaryotic cell membranes, which may be a powerful tool for a variety of drug delivery applications. However, the mechanisms involved in shock wave-mediated membrane permeabilization are still poorly understood. In this study, the effects on both the permeability and the ultrastructural features of two human cell lineages were investigated after the application of underwater shock waves in vitro. Scanning Electron Microscopy of cells derived from a human embryo kidney (HEK)-293 and Michigan Cancer Foundation (MCF)-7 cells, an immortalized culture derived from human breast adenocarcinoma, showed a small amount of microvilli (as compared to control cells), the presence of hole-like structures, and a decrease in cell size after shock wave exposure. Interestingly, these effects were accompanied by the permeabilization of acid and macromolecular dyes and gene transfection. Trypan blue exclusion assays indicated that cell membranes were porated during shock wave treatment but resealed after a few seconds. Deformations of the cell membrane lasted for at least 5 min, allowing their observation in fixed cells. For each cell line, different shock wave parameters were needed to achieve cell membrane poration. This difference was correlated to successful gene transfection by shock waves. Our results demonstrate, for the first time, that shock waves induce transient micro- and submicrosized deformations at the cell membrane, leading to cell transfection and cell survival. They also indicate that ultrastructural analyses of cell surfaces may constitute a useful way to match the use of shock waves to different cells and settings.

The Synergistic Effect between Electrical and Chemical Factors in Plasma Gene/Molecule-Transfection

NASA Astrophysics Data System (ADS)

Jinno, Masafumi

2016-09-01

This study has been done to know what kind of factors in plasma and processes on cells promote plasma gene/molecule transfection. We have discovered a new plasma source using a microcapillary electrode which enables high transfection efficiency and high cell survivability simultaneously. However, the mechanism of the transfection by plasma was not clear. To clarify the transfection mechanisms by micro plasma, we focused on the effects of electrical (current, charge, field, etc.) and chemical (radicals, RONS, etc.) factors generated by the micro plasma and evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones. At first, the necessity of the electrical factors was estimated by the laser produced plasma (LPP). Mouse L-929 fibroblast cell was cultured on a 96-well plate or 12-well micro slide chamber. Plasmids pCX-EGFP in Tris-EDTA buffer was dropped on the cells and they were exposed to the capillary discharge plasma (CDP) or the LPP. In the case of the CDP, the plasma was generated between the tip of the capillary electrode and the cells so that both electrical and chemical factors were supplied to the cells. In this setup, about 20% of average transfection efficiency was obtained. In the case of the LPP, the plasma was generated apart from the cells so that electrical factors were not supplied to the cells. In this setup, no transfection was observed. These results show that the electrical factors are necessary for the plasma gene transfection. Next, the necessity of the chemical factors was estimated the effect of catalase to remove H2O2 in CDP. The transfection efficiency decreased to 0.4 by scavenging H2O2 with catalase. However, only the solution of H2O2 caused no gene transfection in cells. These results shows that H2O2 is important species to cause gene/molecule transfection but still needs a synergistic effect with electrical or other chemical factors. This work was partly supported by Grants-in-Aid for Scientific Research (25108509 and 15H00896) from JSPS and a grant from Ehime University. The plasmids are prepared by ADRES Shigenobu of Ehime University.

Electrosonic ejector microarray for drug and gene delivery.

PubMed

Zarnitsyn, Vladimir G; Meacham, J Mark; Varady, Mark J; Hao, Chunhai; Degertekin, F Levent; Fedorov, Andrei G

2008-04-01

We report on development and experimental characterization of a novel cell manipulation device-the electrosonic ejector microarray-which establishes a pathway for drug and/or gene delivery with control of biophysical action on the length scale of an individual cell. The device comprises a piezoelectric transducer for ultrasound wave generation, a reservoir for storing the sample mixture and a set of acoustic horn structures that form a nozzle array for focused application of mechanical energy. The nozzles are micromachined in silicon or plastic using simple and economical batch fabrication processes. When the device is driven at a particular resonant frequency of the acoustic horn structures, the sample mixture of cells and desired transfection agents/molecules suspended in culture medium is ejected from orifices located at the nozzle tips. During sample ejection, focused mechanical forces (pressure and shear) are generated on a microsecond time scale (dictated by nozzle size/geometry and ejection velocity) resulting in identical "active" microenvironments for each ejected cell. This process enables a number of cellular bioeffects, from uptake of small molecules and gene delivery/transfection to cell lysis. Specifically, we demonstrate successful calcein uptake and transfection of DNA plasmid encoding green fluorescent protein (GFP) into human malignant glioma cells (cell line LN443) using electrosonic microarrays with 36, 45 and 50 mum diameter nozzle orifices and operating at ultrasound frequencies between 0.91 and 0.98 MHz. Our results suggest that efficacy and the extent of bioeffects are mainly controlled by nozzle orifice size and the localized intensity of the applied acoustic field.

Functional MUC4 suppress epithelial-mesenchymal transition in lung adenocarcinoma metastasis.

PubMed

Gao, Liuwei; Liu, Jun; Zhang, Bin; Zhang, Hua; Wang, Daowei; Zhang, Tiemei; Liu, Yang; Wang, Changli

2014-02-01

The mucin MUC4 is a high molecular weight membrane-bound transmembrane glycoprotein that is frequently detected in invasive and metastatic cancer. The overexpression of MUC4 is associated with increased risks for several types of cancer. However, the functional role of MUC4 is poorly understood in lung adenocarcinoma. Using antisense-MUC4-RNA transfected adenocarcinoma cells, we discovered that the loss of MUC4 expression results in epithelial-mesenchymal transition (EMT). We found morphological alterations and the repression of the epithelial marker E-cadherin in transfected cells. Additionally, the loss of MUC4 caused the upregulation of the mesenchymal marker vimentin compared to control cells. Using a MUC4-knockdown versus control LTEP xenograft mice model (129/sv mice), we also found that EMT happened in lung tissues of MUC4-knockdown-LTEP xenograft mice. Moreover, antisense-MUC4-RNA transfected cells had a significantly increased cellular migration ability in vitro. The loss of MUC4 also occurred in lung adenocarcinoma patients with lymph node metastases. We further investigated MUC4 and found that it plays a critical role in regulating EMT by modulating β-catenin. Taken together, our study reveals a novel role for MUC4 in suppressing EMT and suggests that the assessment of MUC4 may function as a prognostic biomarker and could be a potential therapeutic target for lung adenocarcinoma metastasis.

Spatially-Selective Membrane Permeabilization Induced by Cell-Solution Electrode Atmospheric Pressure Plasma Irradiation

NASA Astrophysics Data System (ADS)

Sasaki, Shota; Hokari, Yutaro; Kanzaki, Makoto; Kaneko, Toshiro

2015-09-01

Gene transfection, which is the process of deliberately introducing nucleic acids into cells, is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure plasma (APP). We have previously reported that the cell membrane permeability, which is closely related with gene transfection, is improved using a cell-solution electrode for generating He-APP. He-APP is irradiated to the solution containing the adherent cells and delivery materials such as fluorescent dyes (YOYO-1) and plasmid DNA (GFP). In case of YOYO-1 delivery, more than 80% of cells can be transferred only in the plasma-irradiated area and the spatially-selective membrane permeabilization is realized by the plasma irradiation. In addition, it is confirmed that plasmid DNA is transfected and the GFP genes are expressed using same APP irradiation system with no obvious cellular damage.

Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo, E-mail: parkjw@knu.ac.kr

Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report anmore » autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.« less

Plasmid DNA Delivery: Nanotopography Matters.

PubMed

Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

PubMed

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

PubMed Central

Deshpande, Mangesh C.; Prausnitz, Mark R.

2007-01-01

Ultrasound and poly(ethylenimine) (PEI) have each separately been shown to increase DNA transfection efficiency. This study tested the hypothesis that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. This in vitro study assessed transfection efficiency of two different DNA plasmids encoding green fluorescent protein and firefly luciferase in two different cells types, a primary culture of human aortic smooth muscle cells and an immortal line of human prostrate cancer cells. We found that ultrasound sonication increased transfection up to 18-fold, DNA complexation with PEI increased transfection up to 90-fold, and the combination of ultrasound and PEI synergistically increased transfection up to 200-fold, which resulted in reporter gene expression by 34% of cells. Kinetic measurements found that the effects of ultrasound alone acted quickly, whereas increased transfection by PEI either alone or in combination with ultrasound strongly benefited from a 4-h incubation with the DNA plasmid after sonication. Although serum reduced absolute expression levels, it did not affect the relative increase in transfection when ultrasound was added to PEI enhancement. Flow cytometry measurements showed that sonication increased intracellular uptake of labelled DNA complexed to PEI by 55% relative to PEI complexation alone. Electrophoresis assay showed no damage to DNA or PEI-DNA complexes after sonication. Overall, these results suggest that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. PMID:17258835

MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

PubMed Central

Huang, Yongyi; Liu, Jianjun; Zhao, Yanhui; Jiang, Lizhen; Huang, Qin

2013-01-01

Sperm abnormalities are one of the main factors responsible for male infertility; however, their pathogenesis remains unclear. The role of microRNAs in the development of sperm abnormalities in infertile men has not yet been investigated. Here, we used human induced pluripotent stem cells to investigate the influence of miR-122 expression on the differentiation of these cells into spermatozoa-like cells in vitro. After induction, mutant miR-122-transfected cells formed spermatozoa-like cells. Flow cytometry of DNA content revealed a significant increase in the haploid cell population in spermatozoa-like cells derived from mutant miR-122-transfected cells as compared to those derived from miR-122-transfected cells. During induction, TNP2 and protamine mRNA and protein levels were significantly higher in mutant miR-122-transfected cells than in miR-122-transfected cells. High-throughput isobaric tags for relative and absolute quantification were used to identify and quantify the different protein expression levels in miR-122- and mutant miR-122-transfected cells. Among all the proteins analyzed, the expression of lipoproteins, for example, APOB and APOA1, showed the most significant difference between the two groups. This study illustrates that miR-122 expression is associated with abnormal sperm development. MiR-122 may influence spermatozoa-like cells by suppressing TNP2 expression and inhibiting the expression of proteins associated with sperm development. PMID:23327642

Effects of AC/DC magnetic fields, frequency, and nanoparticle aspect ratio on cellular transfection of gene vectors

NASA Astrophysics Data System (ADS)

Ford, Kris; Mair, Lamar; Fisher, Mike; Rowshon Alam, Md.; Juliano, Rudolph; Superfine, Richard

2008-10-01

In order to make non-viral gene delivery a useful tool in the study and treatment of genetic disorders, it is imperative that these methodologies be further refined to yield optimal results. Transfection of magnetic nanoparticles and nanorods are used as non-viral gene vectors to transfect HeLa EGFP-654 cells that stably express a mutated enhanced green fluorescent protein (EGFP) gene. We deliver antisense oligonucleotides to these cells designed to correct the aberrant splicing caused by the mutation in the EGFP gene. We also transfect human bronchial endothelial cells and immortalized WI-38 lung cells with pEGFP-N1 vectors. To achieve this we bind the genes to magnetic nanoparticles and nanorods and introduce magnetic fields to effect transfection. We wish to examine the effects of magnetic fields on the transfection of these particles and the benefits of using alternating (AC) magnetic fields in improving transfection rates over direct (DC) magnetic fields. We specifically look at the frequency dependence of the AC field and particle aspect ratio as it pertains to influencing transfection rate. We posit that the increase in angular momentum brought about by the AC field and the high aspect ratio of the nanorod particles, is vital to generating the force needed to move the particle through the cell membrane.

Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway.

PubMed

Wang, Hao; Jin, Weitao; Li, Haibin

2018-02-20

The present study investigated the molecular mechanisms underlying the 4A > C and -349C > T single nucleotide polymorphisms (SNPs) in bone morphogenetic protein receptor type IA (BMPR-IA) gene, which significantly associated with the occurrence and the extent of ossification of the posterior longitudinal ligament (OPLL) in the cervical spine. The SNPs in BMPR-IA gene were genotyped, and the association with the occurrence and severity of OPLL were evaluated in 356 OPLL patients and 617 non-OPLL controls. In stably transfected mouse embryonic mesenchymal stem cells (C3H10T1/2), the expression levels of the BMPR-IA gene and Smad4 protein as well as phosphorylated Smad1/5/8 were detected by Western blotting. In addition, the alkaline phosphatase (ALP) and osteocalcin (OC) activity of osteogenesis specificity protein was assessed using the ALP quantitation and osteocalcin radioimmunoassay kit, respectively. The 4A > C and the -349C > T polymorphisms of BMPR-IA gene were significantly associated with the development of OPLL in the cervical spine. The C allele type in 4A > C polymorphism significantly increases the occurrence and the extent of OPLL. The T allele type in -349C > T polymorphism significantly increases the susceptibility to OPLL, but not the extent of OPLL. The current results further validate our previous observations. The expression levels of BMPR-IA gene were significantly increased in pcDNA3.1/BMPR-IA (mutation type, MT -349C > T; MT 4A > C; MT -349C > T and 4A > C) vector-transfected C3H10T1/2 cells compared to the wild type (WT) vector-transfected cells. The levels of phosphorylated Smad1/5/8 and ALP activity were significantly increased in pcDNA3.1/BMPR-IA (MT -349C > T) vector-transfected C3H10T1/2 cells compared to the WT vector-transfected cells. However, no significant differences were observed in the protein levels of phosphorylated Smad1/5/8 and the ALP activity between MT A/C and WT vector-transfected cells. In addition, no significant differences were observed in the Smad4 protein levels among the experimental groups, as well as in the OC activity between WT vector-transfected and MT C/T, MT A/C, MT C/T and MT A/C vector-transfected cells. Our results suggest that Smad signaling pathway may play important roles in the pathological process of OPLL induced by SNPs in BMPR-IA gene. These results will help to clarify the molecular mechanisms underlying the SNP and gene susceptibility to OPLL.

Femtosecond-laser assisted cell reprogramming

NASA Astrophysics Data System (ADS)

Breunig, Hans Georg; Uchugonova, Aisada; Batista, Ana; König, Karsten

2017-02-01

Femtosecond-laser pulses can assist to transfect cells by creating transient holes in the cell membrane, thus making them temporarily permeable for extraneous genetic material. This procedure offers the advantage of being completely "virus free" since no viruses are used for the delivery and integration of gene factors into the host genome and, thereby, avoiding serious side effects which so far prevent clinical application. Unfortunately, focusing of the laser radiation onto individual cell membranes is quite elaborate and time consuming. Regarding these obstacles, we briefly review two optical setups for fast, efficient and high throughput laser-assisted cell transfection based on femtosecond laser pulse excitation. The first setup aims at assisting the transfection of adherent cells. It comprises of a modified laser-scanning microscope with beamshaping optics as well as home-made software to automate the detection, targeting and laser-irradiation process. The second setup aims at laser-assisted transfection of non-adherent cells in suspension which move in a continuous flow through the laser focus region. The setup allows to address a large number of cells, however, with much lower transfection efficiency than the individual-cell targeting approach.

A reverse genetics system for enterovirus D68 using human RNA polymerase I.

PubMed

Pan, Minglei; Gao, Shuai; Zhou, Zhenwei; Zhang, Keke; Liu, Sihua; Wang, Zhiyun; Wang, Tao

2018-05-17

Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5'-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.

Regulated release of a novel non-viral gene delivery vector from electrospun coaxial fiber mesh scaffolds

NASA Astrophysics Data System (ADS)

Saraf, Anita

The development of novel strategies for tissue engineering entails the evolution of biopolymers into multifunctional constructs that can support the proliferation of cells and stimulate their differentiation into functional tissues. With that in mind, biocompatible polymers were fabricated into a novel gene delivery agent as well as three dimensional scaffolds that act as reservoirs and controlled release constructs. To fabricate a novel gene delivery agent a commercially available cationic polymer, poly(ethylenimine), PEI, was chemically conjugated to a ubiquitous glycosaminoglycan, hyaluronic acid (HA). The novel polymer, PEI-HA, had significantly reduced toxicity and improved transfection efficiency with multipotent human mesenchymal stem cells. This transfection efficiency could further be modulated by changing the concentration of sodium chloride and temperature used to assemble PEI-HA/DNA complexes. To facilitate the regulated delivery of these complexes in the context of tissue engineering, an emerging technology for scaffold fabrication, coaxial electrospinning was adapted to include PEI-HA and plasmid DNA within the scaffold fibers. Initially, a factorial design was employed to assess the influence of processing parameters in the absence of gene delivery vectors and plasmids. The study elucidated the role of sheath polymer concentration and core polymer concentration and molecular weight and the presence of sodium chloride on fiber diameters and morphologies. Subsequently, PEI-HA and plasmid DNA were entrapped within the sheath and core compartments of these fibers and the influence of processing parameters was assessed in the context of fiber diameter, release kinetics and transfection efficiency over a period of 60 days. The release of PEI-HA was found to be dependent upon the loading dose of the vector and plasmid. However, the transfection efficiency correlated to the core polymer properties, concentration and molecular weight. The processing parameters could modulate cell transfection for up to 21 days and continue to transfect cells for up to 60 days. Thus, scaffolds with tunable release kinetics and transfection efficiencies can be fabricated using coaxial electrospinning, which can further be used for tissue engineering and gene delivery applications.

Evaluation of cationic liposomes composed of an amino acid-based lipid for neuronal transfection.

PubMed

Obata, Yosuke; Ciofani, Gianni; Raffa, Vittoria; Cuschieri, Alfred; Menciassi, Arianna; Dario, Paolo; Takeoka, Shinji

2010-02-01

We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C(16)) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C(16) liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP-positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C(16) lipoplexes, the proportion of GFP-positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C(16), as a model cationic amino acid-based lipid, has a high capability as a gene carrier, even for neuronal transfection. In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases. Copyright 2010 Elsevier Inc. All rights reserved.

Reduced N-Type Ca2+ Channels in Atrioventricular Ganglion Neurons Are Involved in Ventricular Arrhythmogenesis.

PubMed

Zhang, Dongze; Tu, Huiyin; Cao, Liang; Zheng, Hong; Muelleman, Robert L; Wadman, Michael C; Li, Yu-Long

2018-01-15

Attenuated cardiac vagal activity is associated with ventricular arrhythmogenesis and related mortality in patients with chronic heart failure. Our recent study has shown that expression of N-type Ca 2+ channel α-subunits (Ca v 2.2-α) and N-type Ca 2+ currents are reduced in intracardiac ganglion neurons from rats with chronic heart failure. Rat intracardiac ganglia are divided into the atrioventricular ganglion (AVG) and sinoatrial ganglion. Ventricular myocardium receives projection of neuronal terminals only from the AVG. In this study we tested whether a decrease in N-type Ca 2+ channels in AVG neurons contributes to ventricular arrhythmogenesis. Lentiviral Ca v 2.2-α shRNA (2 μL, 2×10 7  pfu/mL) or scrambled shRNA was in vivo transfected into rat AVG neurons. Nontransfected sham rats served as controls. Using real-time single-cell polymerase chain reaction and reverse-phase protein array, we found that in vivo transfection of Ca v 2.2-α shRNA decreased expression of Ca v 2.2-α mRNA and protein in rat AVG neurons. Whole-cell patch-clamp data showed that Ca v 2.2-α shRNA reduced N-type Ca 2+ currents and cell excitability in AVG neurons. The data from telemetry electrocardiographic recording demonstrated that 83% (5 out of 6) of conscious rats with Ca v 2.2-α shRNA transfection had premature ventricular contractions ( P <0.05 versus 0% of nontransfected sham rats or scrambled shRNA-transfected rats). Additionally, an index of susceptibility to ventricular arrhythmias, inducibility of ventricular arrhythmias evoked by programmed electrical stimulation, was higher in rats with Ca v 2.2-α shRNA transfection compared with nontransfected sham rats and scrambled shRNA-transfected rats. A decrease in N-type Ca 2+ channels in AVG neurons attenuates vagal control of ventricular myocardium, thereby initiating ventricular arrhythmias. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Effects of BDNF-Transfected BMSCs on Neural Functional Recovery and Synaptophysin Expression in Rats with Cerebral Infarction.

PubMed

Zhang, Yongming; Qiu, Binghui; Wang, Jinbiao; Yao, Yi; Wang, Chunlin; Liu, Jiachuan

2017-07-01

The purpose of this study was to investigate the effects of brain-derived neurotrophic factor (BDNF)-transfected bone marrow mesenchymal stem cells (BMSCs) on neural functional recovery and synaptophysin expression in rats with cerebral infarction (CI). A total of 120 healthy Sprague Dawley rats were randomly divided into sham group, control group, and model group. Craniotomy was conducted and neurological function defect scoring was used to verify the model. BDNF containing recombinant plasmid was transfected into rat BMSCs, which was verified by flow cytometry and Western Blot. After injection of the transfected BMSCs, neural functional recovery of the CI rats and synaptophysin expression were measured. After the CI rat model was established, magnetic resonance (MR) imaging, 2, 3, 5- triphenyl tetrazolium chloride (TTC) staining, and the neurological function defect scoring determined the success of the model. CD34 (-), CD45 (-), CD29 (+), and CD90 (+) cells detected showed that the obtained BMSCs have high purity. BDNF protein was highly expressed in the BMSCs successfully transfected with the recombinant plasmid. Balance beam walking score, rotating bar walking score, and screen test score were significantly lower, while synaptophysin expression was higher in the BDNF model group than those in the non-BDNF model group and sham group with time extension. BDNF can increase synaptic plasticity and neurogenesis and have a promotional role in neural functional recovery and synaptophysin expression in rats with CI. BDNF-transfected BMSCs may therefore have better treatment efficacy for CI clinically.

Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection.

PubMed

Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

2014-01-01

In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

Structural characterization of a new lipid/DNA complex showing a selective transfection efficiency in ovarian cancer cells

NASA Astrophysics Data System (ADS)

Caracciolo, G.; Pozzi, D.; Caminiti, R.; Congiu Castellano, A.

2003-04-01

We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties.

Microvalve controlled multi-functional microfluidic chip for divisional cell co-culture.

PubMed

Li, Rui; Zhang, Xingjian; Lv, Xuefei; Geng, Lina; Li, Yongrui; Qin, Kuiwei; Deng, Yulin

2017-12-15

Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP + ) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications. Copyright © 2017. Published by Elsevier Inc.

Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

NASA Astrophysics Data System (ADS)

Das, Debobrato

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

PubMed

Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

2015-08-07

Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer. The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Inhibition of IGF1-R overcomes IGFBP7-induced chemotherapy resistance in T-ALL.

PubMed

Bartram, Isabelle; Erben, Ulrike; Ortiz-Tanchez, Jutta; Blunert, Katja; Schlee, Cornelia; Neumann, Martin; Heesch, Sandra; Baldus, Claudia D

2015-10-08

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with the need for treatment optimization. Previously, high expression of Insulin-like growth factor binding protein 7 (IGFBP7), a member of the IGF system, was identified as negative prognostic factor in adult T-ALL patients. Since aberrant IGFBP7 expression was observed in a variety of neoplasia and was relevant for prognosis in T-ALL, we investigated the functional role of IGFBP7 in Jurkat and Molt-4 cells as in vitro models for T-ALL. Jurkat and Molt-4 cells were stably transfected with an IGFBP7 over-expression vector or the empty vector as control. Proliferation of the cells was assessed by WST-1 assays and cell cycle status was measured by flow-cytometry after BrDU/7-AAD staining. The effect of IGFBP7 over-expression on sensitivity to cytostatic drugs was determined in AnnexinV/7-AAD assays. IGF1-R protein expression was measured by Western Blot and flow-cytometric analysis. IGF1-R associated gene expression profiles were generated from microarray gene expression data of 86 T-ALL patients from the Microarrays Innovations in Leukemia (MILE) multicenter study. IGFBP7-transfected Jurkat cells proliferated less, leading to a longer survival in a nutrient-limited environment. Both IGFBP7-transfected Jurkat and Molt-4 cells showed an arrest in the G0/G1 cell cycle phase. Furthermore, Jurkat IGFBP7-transfected cells were resistant to vincristine and asparaginase treatment. Surface expression and whole protein measurement of IGF1-R protein expression showed a reduced abundance of the receptor after IGFBP7 transfection in Jurkat cells. Interestingly, combination of the IGF1-R inhibitor NPV-AEW541 restored sensitivity to vincristine in IGFBP7-transfected cells. Additionally, IGF1-R associated GEP revealed an up-regulation of important drivers of T-ALL pathogenesis and regulators of chemo-resistance and apoptosis such as NOTCH1, BCL-2, PRKCI, and TP53. This study revealed a proliferation inhibiting effect of IGFBP7 by G0/G1 arrest and a drug resistance-inducing effect of IGFBP7 against vincristine and asparaginase in T-ALL. These results provide a model for the previously observed association between high IGFBP7 expression and chemotherapy failure in T-ALL patients. Since the resistance against vincristine was abolished by IGF1-R inhibition, IGFBP7 could serve as biomarker for patients who may benefit from therapies including IGF1-R inhibitors in combination with chemotherapy.

KGFR as a possible therapeutic target in middle ear cholesteatoma.

PubMed

Yamamoto-Fukuda, Tomomi; Akiyama, Naotaro; Shibata, Yasuaki; Takahashi, Haruo; Ikeda, Tohru; Kohno, Michiaki; Koji, Takehiko

2014-11-01

We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.

[The crosstalk between canonical and noncanonical Wnt signaling pathway in osteoblast differentiation of periodontal ligament stem cells in inflammatory microenvironments].

PubMed

Liu, N; Shi, H G; Zhang, W; Gu, B

2016-11-09

Objective: To investigate the crosstalk between canonical Wnt/β-catenin and noncanonical Wnt/Ca 2+ pathway in osteoblast differentiation process of periodontal ligament stem cell (PDLSC) in inflammatory microenvironments. Methods: PDLSCs were obtained from human healthy individuals(H-PDLSC) and patients with periodontitis(P-PDLSC). The H/P-PDLSCs were transfected with β-catenin siRNA. Cell morphology was observed under fluorescent microscope and transfection efficiency was easured by Western blotting after transfection of PDLSC. The mRNA expressions of Runt-related transcription factor 2(Runx2), β-catenin and nemo like kinase(NLK) were detected by real time PCR, the protein expressions of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and NLK were examined by Western blotting and the CaMK Ⅱ was observed by immunofluorescence staining, respectively. Results: The β-catenin expressions in H/P-PDLSCs were inhibited specifically and efficiently by treatment of β-catenin-siRNA for 24 h. After a 3-day-osteogenic process, results of real-time quantitative PCR showed that the Runx2 mRNA expression in P-PDLSC siRNA β-catenin transfected group(4.553 ± 0.659) was significantly higher than that in P-PDLSC empty plasmid control group(1.918 ± 0.315) ( P= 0.000). A similar trend was observed in the NLK mRNA expression tests(7.341 ± 1.331 vs. 5.664 ± 0.792) ( P= 0.030). Accordingly, the protein expression levels of CaMK Ⅱ, NLK were higher in P-PDLSC siRNA β-catenin transfected group than that in P-PDLSC empty plasmid control group in osteogenic differentiation condition for 3 days. CaMKⅡ was more strongly induced in P-PDLSC siRNA β-catenin group than that in P-PDLSC empty plasmid control group after PDLSC cultured in osteogenic medium for 3 days. Conclusions: Both canonical Wnt/β-catenin and noncanonical Wnt/Ca 2+ pathway could regulate the osteogenic differentiation potential of P-PDLSC. Suppression of β-catenin by siRNA promoted osteogenic differentiation via increasing noncanonical Wnt signaling pathway of PDLSC in inflammatory microenvironments.

Generation of CTL responses against pancreatic cancer in vitro using dendritic cells co-transfected with MUC4 and survivin RNA.

PubMed

Chen, Jiang; Guo, Xiao-Zhong; Li, Hong-Yu; Liu, Xu; Ren, Li-Nan; Wang, Di; Zhao, Jia-Jun

2013-09-23

Pancreatic cancer (PC) is one of the most devastating human malignancies without effective therapies. Tumor vaccine based on RNA-transfected dendritic cells (DCs) has emerged as an alternative therapeutic approach for a variety of human cancers including advanced PC. In the present study we compared the cytotoxic T lymphocyte (CTL) responses against PC cells in vitro, which were induced by DCs co-transfected with two mRNAs of tumor associated-antigens (TAA) MUC4 and survivin, versus DCs transfected with a single mRNA encoding either MUC4 or survivin. DCs co-transfected with two TAA mRNAs were found to induce stronger CTL responses against PC target cells in vitro, compared with the DCs transfected with a single mRNA. Moreover, the antigen-specific CTL responses were MHC class I-restricted. These results provide an experimental foundation for further clinical investigations of DC vaccines encoding multiple TAA epitopes for metastatic PC. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation.

PubMed

Lemmon, Jason C M; McFarland, Ryan J; Rybicka, Joanna M; Balce, Dale R; McKeown, Kyle R; Krohn, Regina M; Matsunaga, Terry O; Yates, Robin M

2011-08-31

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies. Copyright © 2011 Elsevier B.V. All rights reserved.

A potential targeting gene vector based on biotinylated polyethyleneimine/avidin bioconjugates.

PubMed

Zeng, Xuan; Sun, Yun-Xia; Zhang, Xian-Zheng; Cheng, Si-Xue; Zhuo, Ren-Xi

2009-08-01

To improve the gene delivery efficiency and safety of non-viral vector in liver cells, avidin, which exhibited good biocompatibility and remarkable accumulation in liver, was bioconjugated with biotinylated polyethylenimine to obtain a novel gene vector. Biotinylated polyethyleneimine/avidin bioconjugate (ABP) was synthesized through grafting biotin to high molecular weight branched polyethylenimine (PEI, 25 kDa) and then bioconjugating with avidin by the biotin-avidin interaction. Physiochemical characteristics of ABP/pDNA complexes were analyzed, and in vitro cytotoxicity and transfection of ABP were also evaluated in HepG2, Hela and 293 T cells by using 25 kDa PEI as the control. It was found that ABP was able to condense pDNA efficiently at N/P ratio of 4. The particle sizes of ABP/pDNA complexes were less than 220 nm, and the average surface charges were around 27 mV at the N/P ratio ranging from 2 to 60. Among three different cell lines, ABP and its DNA complexes demonstrated much lower cytotoxicity and higher transfection efficacy in HepG2 cells as compared with 25 kDa PEI. ABP presented higher transfection efficacy and safety in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells.

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

PubMed Central

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-01-01

AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators. PMID:12632483

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray.

PubMed

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-03-01

To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

Establishment of human hair follicle mesenchymal stem cells with overexpressed human hepatocyte growth factor.

PubMed

Zhou, Dan; Cheng, Hongjing; Liu, Jinyu; Zhang, Lei

2017-06-01

Chronic liver disease has become a major health problem that causes serious damage to human health. Since the existing treatment effect was not ideal, we need to seek new treatment methods. We utilized the gene recombination technology to obtain the human hair mesenchymal stem cells which overexpression of human hepatocyte growth factor (hHGF). Furthermore, we verified the property of transfected cells through detecting surface marker by flow cytometry. We show here establishment of the hHGF-overexpressing lentivirus vector, and successfully transfection to human hair follicle mesenchymal stem cells. The verified experiments could demonstrate the human hair follicle mesenchymal stem cells which have been transfected still have the properties of stem cells. We successfully constructed human hair follicle mesenchymal stem cells which overexpression hHGF, and maintain the same properties compared with pro-transfected cells.

miR-138 overexpression is more powerful than hTERT knockdown to potentiate apigenin for apoptosis in neuroblastoma in vitro and in vivo

PubMed Central

Chakrabarti, Mrinmay; Banik, Naren L.; Ray, Swapan K.

2013-01-01

Decrease in expression of the tumor suppressor microRNA-138 (miR-138) correlates well with an increase in telomerase activity in many human cancers. The ability of almost all human cancer cells to grow indefinitely is dependent on presence of telomerase activity. The catalytic component of human telomerase reverse transcriptase (hTERT) regulates telomerase activity in most of the human cancers including malignant neuroblastoma. We observed an indirect increase in the expression of miR-138 after the transfection with hTERT short hairpin RNA (shRNA) plasmid in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines. Transfection with hTERT shRNA plasmid followed by treatment with the flavonoid apigenin (APG) further increased expression of miR-138. Direct transfection with miR-138 mimic was more powerful than transfection with hTERT shRNA plasmid in potentiating efficacy of APG for decreasing cell viability and colony formation capability of both cell lines. Upregulation of miR-138 was also more effective than down regulation of hTERT in enhancing efficacy of APG for induction of apoptosis in malignant neuroblastoma cells in vitro and in vivo. We delineated that apoptosis occurred with induction of molecular components of the extrinsic and intrinsic pathways in SK-N-DZ and SK-N-BE2 cells both in vitro and in vivo. In conclusion, these results demonstrate that direct miR-138 overexpression is more powerful than hTERT down regulation in enhancing pro-apoptotic effect of APG for controlling growth of human malignant neuroblastoma in cell culture and animal models. PMID:23562653

Functional Geno,ic Analysis of Breast Cancer Cell Tumorigenicity Using a Noval Gene Silencing Resource

DTIC Science & Technology

2006-04-01

Fig. 2B). In addition, luciferase assay on cells co-transfected with constructs expressing firefly and renilla luciferase genes showed a significant...positive cells. (C) BT474 cells were co-transfected with pGL3 plasmid expressing firefly luciferase, pRL plasmid expressing renilla luciferase, and...genes Per1 (A) and Bmal1 (B). BT474 cells were transfected with Per1 (A) and Bmal1 (B) firefly luciferase reporters, pRL plasmid expressing renilla

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

PubMed

Blasig, I E; Giese, H; Schroeter, M L; Sporbert, A; Utepbergenov, D I; Buchwalow, I B; Neubert, K; Schönfelder, G; Freyer, D; Schimke, I; Siems, W E; Paul, M; Haseloff, R F; Blasig, R

2001-09-01

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature. Copyright 2001 Academic Press.

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

PubMed

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

PubMed

Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively. (1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).

[Killing effects of PWZL plasmid-mediated double suicide gene on human lens epithelium cells].

PubMed

Yan, Xiao-ran; Wu, Hong; Yu, Hai-tao; Wang, Xiu; Zhang, Yu

2008-04-01

To investigate the killing efficiency of PWZL plasmid-mediated herpes simplex virus-thymidine kinase (TK) and E. coli cytosine deaminase (CD) on human lens epithelium cells followed by the treatment of prodrugs. PWZL plasmid was used as a vehicle, to transduce double suicide genes into the human lens epithelium in vitro, then the cells were treated with fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell growth of the lens epithelium cells was observed by light microscope. MTT analysis was used to estimate the cell survival rate and the bystander effect was analyzed simultaneously. The significance of difference between each group was treated by statistical tests. The CD and TK gene could be joined into PWZL plasmid successfully, and did not have any special effect on normal cells. There was no significant difference in cell viability between CD-TK transfected cells and control cells. Cell viability in cells treated with prodrugs was decreased in a time-dependent manner. At the end of the experiment, cell viability was lowest in GCV 10 mg/L +5-FC 60 mg/L group, GCV 10 mg/L + 5-FC 100 mg/L group and GCV 100 mg/L + 5-FC 100 mg/L group. There were no significant differences between these three groups (X2 = 1.25 , P > 0.01). Analysis of bystander effect indicated that the cell viability in GCV 100 mg/L + 5-FC 100 mg/L group and GCV 10 mg/L +5-FC 60 mg/L group was significantly lower than that in the controls (t = 10.26, 13.16; P < 0.01). PWZL plasmid can transfect the CD and TK genes into lens epithelium cells successfully and efficiently. CD and TK genes can be expressed steadily. Transfection of double suicide gene reduces the dosage of prodrugs required for killing cells. The combination of 5-FC with GCV shows the greatest killing effect and also has the bystander effect.

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

PubMed Central

Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

2013-01-01

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

An integrated cell culture lab on a chip: modular microdevices for cultivation of mammalian cells and delivery into microfluidic microdroplets.

PubMed

Hufnagel, Hansjörg; Huebner, Ansgar; Gülch, Carina; Güse, Katharina; Abell, Chris; Hollfelder, Florian

2009-06-07

We present a modular system of microfluidic PDMS devices designed to incorporate the steps necessary for cell biological assays based on mammalian tissue culture 'on-chip'. The methods described herein include the on-chip immobilization and culturing of cells as well as their manipulation by transfection. Assessment of cell viability by flow cytrometry suggests low attrition rates (<3%) and excellent growth properties in the device for up to 7 days for CHO-K1 cells. To demonstrate that key procedures from the repertoire of cell biology are possible in this format, transfection of a reporter gene (encoding green fluorescent protein) was carried out. The modular design enables efficient detachment and recollection of cells and allows assessment of the success of transfection achieved on-chip. The transfection levels (20%) are comparable to standard large scale procedures and more than 500 cells could be transfected. Finally, cells are transferred into microfluidic microdoplets, where in principle a wide range of subsequent assays can be carried out at the single cell level in droplet compartments. The procedures developed for this modular device layout further demonstrate that commonly used methods in cell biology involving mammalian cells can be reliably scaled down to allow single cell investigations in picolitre volumes.

A transfected cell model for the renal toxin transporter, rOCT2.

PubMed

Pan, B F; Sweet, D H; Pritchard, J B; Chen, R; Nelson, J A

1999-02-01

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Use of inert gas jets to measure the forces required for mechanical gene transfection

PubMed Central

2012-01-01

Background Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. Methods We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. Results The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 ± 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. Conclusions The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods. PMID:22963645

Temporal Control of Transforming Growth Factor (TGF) - Betal Expression on Mammary Cell Multistep Transformation

DTIC Science & Technology

2001-10-01

tu- vation of transcription and deregulated cell mors and may eventually regress through growth (18). The importance of APC and [- cat - apoptosis (25...receptors, fibrosarcoma cells transfected to express 10ng/ml TPRII [621, ALK-1 [63], and endoglin [64], and one of its TGF-131 in vitro are unable to

Inorganic nanoparticles for transfection of mammalian cells and removal of viruses from aqueous solutions.

PubMed

Link, Nils; Brunner, Tobias J; Dreesen, Imke A J; Stark, Wendelin J; Fussenegger, Martin

2007-12-01

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.

Synergistic effect of electrical and chemical factors on endocytosis in micro-discharge plasma gene transfection

NASA Astrophysics Data System (ADS)

Jinno, M.; Ikeda, Y.; Motomura, H.; Isozaki, Y.; Kido, Y.; Satoh, S.

2017-06-01

We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity; however, the mechanism underlying the method is still unknown. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. The electric field threshold required for transfection was approximately 1 kV m-1 in our MDP system. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage.

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

PubMed

Kondo, Norihiko; Ishii, Yasuyuki; Son, Aoi; Sakakura-Nishiyama, Junko; Kwon, Yong-Won; Tanito, Masaki; Nishinaka, Yumiko; Matsuo, Yoshiyuki; Nakayama, Toshinori; Taniguchi, Masaru; Yodoi, Junji

2004-03-29

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

A perchlorate sensitive iodide transporter in frogs

PubMed Central

Carr, Deborah L.; Carr, James A.; Willis, Ray E.; Pressley, Thomas A.

2008-01-01

Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater 125I uptake than sham-transfected control cells. The uptake in carrier-transfected cells was significantly inhibited in the presence of perchlorate, a competitive inhibitor of mammalian Na+/iodide symporter. Tissue distributions of the sequence were also consistent with a role in iodide uptake. The mRNA encoding the carrier was found to be expressed in the thyroid gland, stomach, and kidney of tadpoles from X. laevis, as well as the bullfrog Rana catesbeiana. The ovaries of adult X. laevis also were found to express the carrier. Phylogenetic analysis suggested that the putative X. laevis iodide transporter is orthologous to vertebrate Na+-dependent iodide symporters. We conclude that the amphibian sequence encodes a protein that is indeed a functional Na+/iodide symporter in Xenopus laevis, as well as Rana catesbeiana. PMID:18275962

AJUBA increases the cisplatin resistance through hippo pathway in cervical cancer.

PubMed

Bi, Lihong; Ma, Feng; Tian, Rui; Zhou, Yanli; Lan, Weiguang; Song, Quanmao; Cheng, Xiankui

2018-02-20

Though LIM-domain protein AJUBA was identified as a putative oncogene, the function and underlying mechanisms of AJUBA in cervical cancer remain largely unknown. Firstly, AJUBA expression was detected via real-time quantitative PCR in patients' samples. Furthermore, Hela and Siha cells were transfected with AJUBA-overexpressing plasmids, and then exposed to cisplatin, the apoptosis was measured by cytometry assay. In addition, the expression of YAP and TAZ was disclosed through western blot assay. Our results revealed that AJUBA expression was significantly higher in the cervical cancer patients resistant to cisplatin treatment compared with cervical cancer patients sensitive to cisplatin treatment. In addition, overall survival time was significantly shorter in the cervical cancer patients with high AJUBA expression compare with those with low AJUBA expression using kaplan-meier analysis. Hela and Siha cells transfected with AJUBA-expressing plasmids exposed to cisplatin treatment had higher survival rate compared with the cells transfected with empty vector control. Mechanistic studies revealed the AJUBA upregulated the downstream targets YAP and TAZ. These results suggest that high AJUBA level enhances cervical cancer cells drug resistance to cisplatin, also associates with decreased patient survival times. Copyright © 2017 Elsevier B.V. All rights reserved.

STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

PubMed

Terra, Silvia R; Cardoso, João Carlos R; Félix, Rute C; Martins, Leo Anderson M; Souza, Diogo Onofre G; Guma, Fatima C R; Canário, Adelino Vicente M; Schein, Vanessa

2015-03-05

Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

[Algal oligosaccharides ameliorate osteoporosis via up-regulation of parathyroid hormone 1-84 and vascular endothelial growth factor].

PubMed

Wang, Li; Wang, Haiya; Fang, Ningyuan

2016-06-01

To determine whether algal oligosac- charide~ affects the levels of parathyroid hormone 1-84 (PTH1-84) and vascular endothelial growth fac- tor (VEGF). An osteoporosis rat model was estab- lished via bilateral ovariectomy. The model rats were fed algal oligosaccharides (molecular weights: 600-1, 200 Da) for 4 months. Bone mineral density (BMD) was then measured. MG-63 human osteo- blastic cells were treated with algal oligosaccha- rides. The expression of PTH1-84 and VEGF was then examined. Oligosaccharide-treated cells were transfected with PTH1-84 short hairpin RNA (shR- NA), VEGF shRNA, and PTH1-84-VEGF small interfer- ing RNA (siRNA). The growth rates were then com- pared between transfected and non-transfected Algal oligosaccharides increased the BMD of the osteoporosis rat model compared with untreated controls (P < 0.05). When MG-63 cells were treated with algal oligosaccharides, the growth rate increased by 25% compared with the control group at day 3 (P < 0.05). In addition, the ex- pression of P.TH84 and VEGF was. enhanced. Con- versey w hen tecells were tranfected with PTH84 shRNA, VEGF shRNA, or PTH1-84-VEGF siR- NA, the growth rate was decreased by 17%, 35% and 70%, respectively, compared with controls at day 3 (P < 0.05). Algal oligosaccharides ameliorate osteoporosis via up-regulation of PTH1-84 and VEGF. Algal oligosaccharides should be developed as a potential drug for osteoporosis treatment.

Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

PubMed

Lang, Annemarie; Neuhaus, Johannes; Pfeiffenberger, Moritz; Schröder, Erik; Ponomarev, Igor; Weber, Yvonne; Gaber, Timo; Schmidt, Michael F G

2014-01-01

Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

PubMed Central

1993-01-01

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer. PMID:8391000

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

PubMed

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro.

PubMed

Parsa, A T; Chi, J H; Hurley, P T; Jeyapalan, S A; Bruce, J N

2001-09-01

Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. Induction of MHC Class I in rat and human glioma cells after HSV TK retroviral gene therapy is a primary effect that is dependent on tyrosine kinase activity. Specific immune responses generated after transfection may represent an important general side effect of gene therapy protocols. Elucidation of the mechanism of immunomodulation after gene therapy will likely yield safer and more effective clinical protocols.

Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer.

PubMed

Behboodi, E; Ayres, S L; Memili, E; O'Coin, M; Chen, L H; Reggio, B C; Landry, A M; Gavin, W G; Meade, H M; Godke, R A; Echelard, Y

2005-01-01

Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT. Goats were derived from two fetal cell lines, each transfected with a transgene expressing a different version of the MSP-1(42) malaria antigen, either glycosylated or non-glycosylated. Two female kids were produced per cell line. Health and growth of these NT animals were monitored and compared with four age-matched control does. There were no differences in birth and weaning weights between NT and control animals. The NT does were bred and produced a total of nine kids. The control does delivered five kids. The NT does expressing the glycosylated antigen lactated only briefly, probably as a result of over-expression of the MSP-1(42) protein. However, NT does expressing the non-glycosylated antigen had normal milk yields and produced the recombinant protein. These data demonstrated that the production of healthy transgenic founder goats by somatic cell NT is readily achievable and that these animals can be used successfully for the production of a candidate Malaria vaccine.

Tailoring charge density and hydrogen bonding of imidazolium copolymers for efficient gene delivery.

PubMed

Allen, Michael H; Green, Matthew D; Getaneh, Hiwote K; Miller, Kevin M; Long, Timothy E

2011-06-13

Conventional free radical polymerization with subsequent postpolymerization modification afforded imidazolium copolymers with controlled charge density and side chain hydroxyl number. Novel imidazolium-containing copolymers where each permanent cation contained one or two adjacent hydroxyls allowed precise structure-transfection efficiency studies. The degree of polymerization was identical for all copolymers to eliminate the influence of molecular weight on transfection efficiency. DNA binding, cytotoxicity, and in vitro gene transfection in African green monkey COS-7 cells revealed structure-property-transfection relationships for the copolymers. DNA gel shift assays indicated that higher charge densities and hydroxyl concentrations increased DNA binding. As the charge density of the copolymers increased, toxicity of the copolymers also increased; however, as hydroxyl concentration increased, cytotoxicity remained constant. Changing both charge density and hydroxyl levels in a systematic fashion revealed a dramatic influence on transfection efficiency. Dynamic light scattering of the polyplexes, which were composed of copolymer concentrations required for the highest luciferase expression, showed an intermediate DNA-copolymer binding affinity. Our studies supported the conclusion that cationic copolymer binding affinity significantly impacts overall transfection efficiency of DNA delivery vehicles, and the incorporation of hydroxyl sites offers a less toxic and effective alternative to more conventional highly charged copolymers.

Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase

PubMed Central

Mendoza, Rhone A; Moody, Emily E; Enriquez, Marlene I; Mejia, Sylvia M; Thordarson, Gudmundur

2011-01-01

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate its role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of the estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of the ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E2), whereas growth hormone plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E2, their proliferation rate was not different from controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E2 treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E2-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Conclusions: increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein. PMID:20974639

Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase.

PubMed

Mendoza, Rhone A; Moody, Emily E; Enriquez, Marlene I; Mejia, Sylvia M; Thordarson, Gudmundur

2011-01-01

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate p38 MAPK's role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E₂), whereas GH plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E₂, their proliferation rate was lower compared to controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E₂ treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E₂-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in the apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein.

Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

NASA Astrophysics Data System (ADS)

Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

1995-07-01

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Up-Regulation of Autophagy in Small Intestine Paneth Cells in Response to Total-Body gamma-Irradiation

DTIC Science & Technology

2009-01-01

standard error of the mean (SEM). Analysis of variance procedures with Tukey post hoc correction examined the existence and nature of temporal trends ...apoptosis. Cell 2006;126:121–134. 20. Yorimitsu T, Klionsky DJ. Eating the enoplasmic reticulum: quality control by autophagy. Trends Cell Biol 2007;17...oxide signaling to iron- regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected

Overexpression of Annexin A1 Suppresses Pro-Inflammatory Factors in PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium

PubMed Central

Kiani-Esfahani, Abbas; Kazemi Sheykhshabani, Sedigheh; Peymani, Maryam; Hashemi, Motahare-Sadat; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2016-01-01

Objective Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). Materials and Methods In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. Results Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. Conclusion ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions. PMID:27540524

Overexpression of Annexin A1 Suppresses Pro-Inflammatory Factors in PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium.

PubMed

Kiani-Esfahani, Abbas; Kazemi Sheykhshabani, Sedigheh; Peymani, Maryam; Hashemi, Motahare-Sadat; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2016-01-01

Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions.

[Expression of OPN gene during different lactation stages in mammary gland of dairy goat and its effect on growth of MCF-7 cell line].

PubMed

Sun, Jie; Luo, Jun; Liu, Jun-Xia; Li, Da-Quan

2009-08-01

To investigate the expression pattern and preliminary function of OPN gene in mammary gland of dairy goat during different lactation stages, using b-actin gene as the internal control, the SYBR Green quantitative real-time PCR (QPCR) analysis was conducted to determine the mRNA expression of OPN gene in mammary gland at the 28th, 60th, 100th, 190th, 270th and 330th day after kidding. Recombinant plasmid of pcDNA3.1-OPN was constructed by inserting the fragment of OPN gene into eukaryotic expression vector pcDNA3.1 and used to transfect the MCF-7 cell line following the restrictive endonuclease cleavage and sequence identification of the target gene segment, the effect of OPN gene on MCF-7 cell proliferation was assessed by MTT analysis. The results indicated that OPN gene exhibited the higher expression level in early (28 d) and late (190 d) lactation stages and the lowest level at dry stage (330 d), which demonstrated a high-low-high-low pattern. There was a significant difference (P < 0. 05) in the proliferation between OPN gene transfected and non-transfected MCF-7 cells, which suggested that the expression of OPN gene could stimulate the proliferation of MCF-7 cells.

Polyethyleneimine-capped silver nanoclusters for microRNA oligonucleotide delivery and bacterial inhibition

PubMed Central

Liang, Jichao; Luo, Ailing; Wang, Lingqian; Zhu, Jing; Xiong, Huayu; Chen, Yong

2017-01-01

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this paper, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were prepared for the purpose of microRNA (miRNA) delivery. The resultant PEI-AgNCs were characterized by a photoluminescence assay and transmission electron microscopy. A cytotoxicity assay showed that PEI-AgNCs exhibit relatively low cytotoxicity. Interestingly, PEI-AgNCs were confirmed to transfect miRNA mimics more effectively than PEI in HepG2 and 293A cells. In this regard, hsa-miR-21 or hsa-miR-221 mimics (miR-21/221m) were transported into HepG2 cells by using PEI-AgNCs. The miR-21/221 expression was determined post-transfection by quantitative real-time polymerase chain reaction. Compared with the negative control, PEI-AgNCs/miR-21/221m groups exhibited higher miR-21/221 levels. In addition, AgNCs endow PEI with stronger antibacterial activity, and this advantage provided PEI-AgNCs the potential to prevent bacterial contamination during the transfection process. Furthermore, we showed that PEI-AgNCs are viable nanomaterials for plain imaging of the cells by laser scanning confocal microscopy, indicating great potential as an ideal fluorescent probe to track the transfection behavior. These results demonstrated that PEI-AgNCs are promising and novel nonviral vectors for gene delivery. PMID:29238194

Aggregation and lack of secretion of most newly synthesized proinsulin in non-beta-cell lines.

PubMed

Zhu, Yong Lian; Abdo, Alexander; Gesmonde, Joan F; Zawalich, Kathleen C; Zawalich, Walter; Dannies, Priscilla S

2004-08-01

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

mTOR inhibition of cardamonin on antiproliferation of A549 cells is involved in a FKBP12 independent fashion.

PubMed

Tang, Ying; Fang, Qi; Shi, Daohua; Niu, Peiguang; Chen, Yaoyao; Deng, Jie

2014-03-18

Cardamonin has previously demonstrated that it had an antiproliferative effect on vascular smooth muscle cells by inhibiting the activity of mammalian target of rapamycin (mTOR). The antiproliferative effect and the possible mechanism of combining with mTOR of cardamonin were investigated on A549 cells. Cell proliferation, cell cycle and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. mTOR and 12 kDa FK506 binding protein (FKBP12) were transfected into A549 cells by Lipofectamine. Western blots were used to examine the mTOR expressions and its activities, and the expressions of 70 kDa ribosomal S6 kinase (p70S6K), FKBP12 and Interleukin-2 (IL-2), respectively. Treated with cardamonin, the proliferation of A549 cells was inhibited. Meanwhile, cell cycle was blocked and DNA synthesis was decreased whereas cell apoptosis was promoted, and the activation of mTOR and p70S6K was decreased by cardamonin. Transfected with mTOR or FKBP12, proliferation of A549 cells was increased. Rapamycin had a similar degree of effect on antiproliferation of both transfected cells. However, the antiproliferative effect of cardamonin on mTOR transfected cells was stronger than that on FKBP12 transfected cells. Both rapamycin and cardamonin decreased the phosphorylation of mTOR and p70S6K in two kinds of transfected cells. Cardamonin had no effect on the expression of FKBP12 and IL-2, whereas the expressions were decreased by rapamycin. Cardamonin inhibited proliferation and induced apoptosis of A549 cells via mTOR. It might directly interact with mTOR independently of binding with FKBP12. Copyright © 2014 Elsevier Inc. All rights reserved.

Attenuation of MUC4 potentiates the anticancer activity of auranofin via regulation of the Her2/Akt/FOXO3 pathway in ovarian cancer cells.

PubMed

Bae, Jun Sang; Lee, Jongsung; Park, Yoonkook; Park, Kyungmoon; Kim, Jung Ryul; Cho, Dong Hyu; Jang, Kyu Yun; Park, See-Hyoung

2017-10-01

Previously, we reported that auranofin induces apoptosis in SKOV3 cells via regulation of the IKKβ/FOXO3 pathway. In the present study, we reveal that the anticancer activity of auranofin in SKOV3 cells could be enhanced by the attenuation of MUC4 through the regulation of the Her2/Akt/FOXO3 pathway. Compared to the control-siRNA, siRNA transfection against MUC4 into SKOV3 cells accelerated the protein degradation of Her2. Under the same conditions, the expression level of phosphorylated Akt was also downregulated leading to an increase of FOXO3 in the nucleus. Notably, auranofin treatment in SKOV3 cells also resulted in the downregulation of the expression levels of both Her2 and phosphorylated Akt. Thus, Her2 was identified as the common molecular target protein by siRNA transfection against MUC4. Western blot analysis of total and nuclear fraction lysates from SKOV3 cells revealed that attenuation of MUC4 combined with auranofin treatment in SKOV3 cells synergistically activated FOXO3 translocation from the cytoplasm to the nucleus through the regulation of the Her2/Akt/FOXO3 pathway. Attenuation of MUC4 by siRNA transfection potentiated the antitumor effect of auranofin which was examined by performing in vitro assays such as WST-1, cell counting, colony formation, TUNEL and Annexin V staining. In addition, western blot analysis of the apoptosis‑related proteins such as PARP1, caspase-3, Bim extra large (EL), Bax and Bcl2 revealed that the attenuation of MUC4 by siRNA transfection potentiates the pro-apoptotic activity of auranofin in SKOV3 cells. Collectively, auranofin could regulate the Her2/Akt/FOXO3 signaling pathway in SKOV3 cells and be used as a potential antitumor agent considering the expression of MUC4 in ovarian cancer patients.

Functional expression of a cattle MHC class II DR-like antigen on mouse L cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraser, D.C.; Craigmile, S.; Campbell, J.D.M.

1996-09-01

Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3*0101, which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the tranfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4{sup +} T cellsmore » from both the original animal used to obtain the genes, and also from an unrelated DRBF3{sup +} heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design. 45 refs., 5 figs., 1 tab.« less

Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors.

PubMed

Duan, Xiaoping; Guan, Hui; Cao, Ying; Kleinerman, Eugenie S

2009-01-01

This study evaluated the therapeutic efficacy of interleukin 12 (IL-12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL-12 gene delivery. MSCs were isolated from murine bone marrow cells. Cells were phenotyped using flow cytometry. Cultured MSCs differentiated into osteocytes and adipocytes using the appropriate media. Freshly isolated MSCs were transfected with adenoviral vectors containing either the beta-galactosidase (Ad:beta-gal) or the IL-12 (Ad:IL-12) gene. Expression of IL-12 was confirmed using reverse transcription polymerase chain reaction. Mice with TC71 Ewing sarcoma tumors were then treated intravenously with MSCs transfected with Ad:beta-gal or Ad:IL-12. Tumors were measured and analyzed by immunohistochemical analysis for expression of IL-12 protein. Expression of both p35 and p40 IL-12 subunits was demonstrated in MSCs transfected in vitro with Ad:IL-12. IL-12 expression was seen in tumors from mice treated with MSCs transfected with Ad:IL-12. Tumor growth was also significantly inhibited compared with that in mice treated with MSCs transfected with Ad:beta-gal. MSCs can be transfected with the IL-12 gene. These transfected cells localize to tumors after intravenous injection and induce local IL-12 protein production and the regression of established tumors. Copyright (c) 2008 American Cancer Society.

Optimization of input parameters of acoustic-transfection for the intracellular delivery of macromolecules using FRET-based biosensors

NASA Astrophysics Data System (ADS)

Yoon, Sangpil; Wang, Yingxiao; Shung, K. K.

2016-03-01

Acoustic-transfection technique has been developed for the first time. We have developed acoustic-transfection by integrating a high frequency ultrasonic transducer and a fluorescence microscope. High frequency ultrasound with the center frequency over 150 MHz can focus acoustic sound field into a confined area with the diameter of 10 μm or less. This focusing capability was used to perturb lipid bilayer of cell membrane to induce intracellular delivery of macromolecules. Single cell level imaging was performed to investigate the behavior of a targeted single-cell after acoustic-transfection. FRET-based Ca2+ biosensor was used to monitor intracellular concentration of Ca2+ after acoustic-transfection and the fluorescence intensity of propidium iodide (PI) was used to observe influx of PI molecules. We changed peak-to-peak voltages and pulse duration to optimize the input parameters of an acoustic pulse. Input parameters that can induce strong perturbations on cell membrane were found and size dependent intracellular delivery of macromolecules was explored. To increase the amount of delivered molecules by acoustic-transfection, we applied several acoustic pulses and the intensity of PI fluorescence increased step wise. Finally, optimized input parameters of acoustic-transfection system were used to deliver pMax-E2F1 plasmid and GFP expression 24 hours after the intracellular delivery was confirmed using HeLa cells.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII.

PubMed

Swiech, Kamilla; Kamen, Amine; Ansorge, Sven; Durocher, Yves; Picanço-Castro, Virgínia; Russo-Carbolante, Elisa M S; Neto, Mário S A; Covas, Dimas T

2011-11-24

Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37 °C. We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34 °C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery.

PubMed

Nam, Joung-Pyo; Kim, Soyoung; Kim, Sung Wan

2018-07-10

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102-128 nm) and positive surface charge (27-34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed better results than poly(CBA-DAH) at 4-fold lower weight ratio. The data of all experiments demonstrate that the synthesized PCDP could be used for efficient gene delivery and could be widely applied. Published by Elsevier B.V.

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

PubMed

Ziraksaz, Zarrintaj; Nomani, Alireza; Ruponen, Marika; Soleimani, Masoud; Tabbakhian, Majid; Haririan, Ismaeil

2013-01-23

Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression. Copyright © 2012 Elsevier B.V. All rights reserved.

Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects.

PubMed

Zong, Haibin; Zhao, Hongxing; Zhao, Yilei; Jia, Jingling; Yang, Libin; Ma, Chao; Zhang, Yang; Dong, Yuzhen

2013-05-15

Schwann cells and neurotrophin-3 play an important role in neural regeneration, but the secretion of neurotrophin-3 from Schwann cells is limited, and exogenous neurotrophin-3 is inactived easily in vivo. In this study, we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes. Results showed that neurotrophin-3 was successfully transfected into Schwann cells, where it was expressed effectively and steadily. A composite of Schwann cells transfected with neurotrophin-3 and poly(lactic-co-glycolic acid) biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects. Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination, promote nerve axonal and myelin regeneration, and delay apoptosis of spinal motor neurons. Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.

Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects

PubMed Central

Zong, Haibin; Zhao, Hongxing; Zhao, Yilei; Jia, Jingling; Yang, Libin; Ma, Chao; Zhang, Yang; Dong, Yuzhen

2013-01-01

Schwann cells and neurotrophin-3 play an important role in neural regeneration, but the secretion of neurotrophin-3 from Schwann cells is limited, and exogenous neurotrophin-3 is inactived easily in vivo. In this study, we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes. Results showed that neurotrophin-3 was successfully transfected into Schwann cells, where it was expressed effectively and steadily. A composite of Schwann cells transfected with neurotrophin-3 and poly(lactic-co-glycolic acid) biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects. Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination, promote nerve axonal and myelin regeneration, and delay apoptosis of spinal motor neurons. Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury. PMID:25206420

[Modulation of TLR-4/MyD88 signaling cascade by miR-21 is involved in airway immunologic dysfunction induced by cold air exposure].

PubMed

Xu, Rui; Huang, Huaping; Han, Zhong; Li, Minchao; Zhou, Xiangdong

2016-01-01

To investigate the role of miR-21 in airway immunologic dysfunction induced by cold air irritation. Immortalized human airway epithelial cell lines BEAS-2B and 16HBE cells were cultured in air-liquid phases. The differential expressions of endogenous miR-21, miR-164, and miR-155 in the cells induced by cold air exposure for different time were detected by real-time PCR. The reporter plasmid containing wild-type or mutated 3'UTR of TLR-4 were constructed and co-transfected into BEAS-2B cells or 16HBE cells together with miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, or miR-21 inhibitor control. Following the transfection, dual luciferase reporter assay was performed to verify the action of miR-21 on TLR-4. miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, and miR-21 inhibitor control were transfected via lipofectamine 2000 in BEAS-2B or 16HBE cells that were subsequently exposed to a temperature at 37 degrees celsius; or cold irritation (30 degrees celsius;), and the protein levels of TLR-4/MyD88 were detected by Western blotting. Cold irritation caused a time- dependent up-regulation of miR-21 in both BEAS-2B and 16HBE cells (P<0.05) without obviously affecting the expressions of miR-164 and miR-155. Dual luciferase reporter assay demonstrated a direct combination of miR-21 and its target protein TLR-4. The synthesis levels of TLR-4/MyD88 protein were decreased in miR-21 mimic group even at a routine culture temperature (P<0.05), as also seen in cells with cold irritation (P<0.05). Treatment with the miR-21 inhibitor partially attenuated cold irritation-induced down-regulation of TLR-4/MyD88 protein (P<0.05). Cold air irritation-induced airway immunologic dysfunction is probably associated with TLR-4/MyD88 down-regulation by an increased endogenic miR-21.

Effective Targeted Gene Delivery to Dendritic Cells via Synergetic Interaction of Mannosylated Lipid with DOPE and BCAT

PubMed Central

Kim, Hee-Kwon; Wei, Huiling; Kulkarni, Aditya; Pogranichniy, Roman M.; Thompson, David H.

2012-01-01

The efficient delivery of plasmids encoding antigenic determinants into dendritic cells (DCs) that control immune response is a promising strategy for rapid development of new vaccines. In this study, we prepared a series of targeted cationic lipoplex based on two synthetic lipid components, mannose-poly(ethylene glycol, MW3000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Mannose-PEG3000-DSPE) and O-(2R-1,2-di-O-(1'Z,9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), that were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for evaluation as non-viral vectors for transgene expression in DCs. First, we optimized the N:P ratio for maximum transfection and then screened the effects of mannose targeting for further enhancement of transfection levels. Our results indicate that efficient delivery of gWIZ GFP plasmid into DCs was observed for mannose compositions of ~10%, whereas low transfection efficiencies were observed with non-targeted formulations. Mannose-targeted lipofectamine complexes also showed high GFP expression levels in DCs relative to non-targeted lipofectamine controls. The best transfection performance was observed using 10 mol % Mannose-PEG3000-DSPE, 60 mol% BCAT, and 30 mol % DOPE, indicating that the most efficient delivery into DCs occurs via synergistic interaction between mannose targeting and acid-labile, fusogenic BCAT:DOPE formulations. Our data suggest that mannose-PEG3000-DSPE:BCAT:DOPE formulations may be effective gene delivery vehicles for the development of DC-based vaccines. PMID:22229467

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses (host range restriction/Fv-1 gene/DNA transfection)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1/sup n/ (NIH-3T3) and two Fv-1/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 geonotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-I cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

PubMed

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

A gene delivery system containing nuclear localization signal: Increased nucleus import and transfection efficiency with the assistance of RanGAP1.

PubMed

Chen, Kang; Guo, Lingling; Zhang, Jiulong; Chen, Qing; Wang, Kuanglei; Li, Chenxi; Li, Weinan; Qiao, Mingxi; Zhao, Xiuli; Hu, Haiyang; Chen, Dawei

2017-01-15

In the present report, a degradable gene delivery system (PAMS/DNA/10NLS) containing nucleus location signal peptide (NLS) was prepared. The agarose gel electrophoresis, particle size and zeta potential of PAMS/DNA/10NLS were similar to those of PAMS/DNA, which proved that NLS did not affect the interaction between PAMS and DNA. PAMS/DNA/10NLS exhibited marked extracellular and intracellular degradation under acidic conditions. The degradation was believed to allow NLS to come into contact with importins easily, which was able to mediate the nucleus import. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. Moreover, the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than PAMS/DNA. Considering that GTPase-activating protein 1 (RanGAP1) was able to activate the endogenous GTPase, which was necessary for NLS-mediated nucleus import, RanGAP1 overexpressed cells (RanGAP1 cells) were produced. This result showed that RanGAP1 cells had higher GTPase activities than normal cells. Both the nucleus import and transfection efficiency of PAMS/DNA/10NLS were markedly higher in RanGAP1 cells than that in normal cells. The in vivo transfection results also showed that the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. These findings showed that PAMS/DNA/10NLS is a promising gene delivery system with the assistance of RanGAP1. The present report describes the increased transfection efficiency of a degradable gene delivery system (PAMS/DNA/10NLS) containing nuclear location signal (NLS) with the assistance of GTPase-activating protein 1 (RanGAP1). The physicochemical properties of PAMS/DNA/10NLS were similar to those of PAMS/DNA. PAMS/DNA/10NLS exhibited great extracellular and intracellular degradations, which might allow NLS to contact with importins easily. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. The transfection of PAMS/DNA/10NLS had less dependence on the breakdown of nuclear envelope. Both the nuclear import and transfection efficiency of PAMS/DNA/10NLS were higher in RanGAP1 overexpressed cells than that in normal cells. Moreover, the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-Ibeta/SET.

PubMed

Anazawa, Yoshio; Nakagawa, Hidewaki; Furihara, Mutsuo; Ashida, Shingo; Tamura, Kenji; Yoshioka, Hiroki; Shuin, Taro; Fujioka, Tomoaki; Katagiri, Toyomasa; Nakamura, Yusuke

2005-06-01

Through genome-wide cDNA microarray analysis coupled with microdissection of prostate cancer cells, we identified a novel gene, prostate collagen triple helix (PCOTH), showing overexpression in prostate cancer cells and its precursor cells, prostatic intraepithelial neoplasia (PIN). Immunohistochemical analysis using polyclonal anti-PCOTH antibody confirmed elevated expression of PCOTH, a 100-amino-acid protein containing collagen triple-helix repeats, in prostate cancer cells and PINs. Knocking down PCOTH expression by small interfering RNA (siRNA) resulted in drastic attenuation of prostate cancer cell growth, and concordantly, LNCaP derivative cells that were designed to constitutively express exogenous PCOTH showed higher growth rate than LNCaP cells transfected with mock vector, suggesting the growth-promoting effect of PCOTH on prostate cancer cell. To investigate the biological mechanisms of this growth-promoting effect, we applied two-dimensional differential gel electrophoresis (2D-DIGE) to analyze the phospho-protein fractions in LNCaP cells transfected with PCOTH. We found that the phosphorylation level of oncoprotein TAF-Ibeta/SET was significantly elevated in LNCaP cells transfected with PCOTH than control LNCaP cells, and these findings were confirmed by Western blotting and in-gel kinase assay. Furthermore, knockdown of endogenous TAF-Ibeta expression by siRNA also attenuated viability of prostate cancer cells as well. These findings suggest that PCOTH is involved in growth and survival of prostate cancer cells thorough, in parts, the TAF-Ibeta pathway, and that this molecule should be a promising target for development of new therapeutic strategies for prostate cancers.

Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells

NASA Astrophysics Data System (ADS)

Wang, Ze; Zhang, Hui

As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

Delivery of RNA and its intracellular translation into protein mediated by SDS-CTAB vesicles: potential use in nanobiotechnology.

PubMed

Russo, Laura; Berardi, Valerio; Tardani, Franco; La Mesa, Camillo; Risuleo, Gianfranco

2013-01-01

Catanionic vesicles are supramolecular aggregates spontaneously forming in water by electrostatic attraction between two surfactants mixed in nonstoichiometric ratios. The outer surface charges allow adsorption to the biomembrane by electrostatic interactions. The lipoplex thus obtained penetrates the cell by endocytosis or membrane fusion. We examined the possible cytotoxic effects and evaluated the transfection efficiency of one vesicle type as compared to known commercial carriers. We show that the individual components of two different vesicles types, CTAB (cetyltrimethylammonium bromide) and DDAB (didodecyldimethylammonium bromide) are detrimental for cell survival. We also assayed the cytotoxicity of SDS-DDAB vesicles and showed dose and time dependency, with the DDAB component being per se extremely cytotoxic. The transfection efficiency of exogenous RNA mediated by SDS-CTAB increases if vesicles assemble in the presence of the reporter RNA; finally, freezing abrogates the transfection ability. The results of our experimental strategy suggest that catanionic vesicles may be adopted in gene therapy and control of antiproliferative diseases.

High throughput RNAi assay optimization using adherent cell cytometry.

PubMed

Nabzdyk, Christoph S; Chun, Maggie; Pradhan, Leena; Logerfo, Frank W

2011-04-25

siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). AoSMC were seeded at a density of 3000-8000 cells/well of a 96 well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

High throughput RNAi assay optimization using adherent cell cytometry

PubMed Central

2011-01-01

Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs. PMID:21518450

Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells.

PubMed

Ma, Y F; Wu, Z H; Gao, M; Loor, J J

2018-06-01

Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H 2 O 2 (600 μM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H 2 O 2 , BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H 2 O 2 . In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H 2 O 2 compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H 2 O 2 compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H 2 O 2 -induced cytotoxicity. The relevance of this kinase in vivo merits further study. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

PubMed Central

Aljubran, Salman A.; Rajanbabu, Venugopal; Bao, Huynh; Mohapatra, Shyam M.; Lockey, Richard; Kolliputi, Narasaiah

2012-01-01

Introduction Pulmonary Arterial Hypertension (PAH) is a progressively devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. The proliferation of cancer cells is mediated by increased expression of Enhancer of Zeste Homolog 2 (EZH2), a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes. However, the role of EZH2 in PAH has not been studied. In this study, it is hypothesized that EZH2 could play a role in the proliferation of PASMCs. Methods In the present study, the expression patterns of EZH2 were investigated in normal and hypertensive mouse PASMCs. The effects of EZH2 overexpression on the proliferation of human PASMCs were tested. PASMCs were transfected with EZH2 or GFP using nucleofector system. After transfection, the cells were incubated for 48 hours at 37°C. Proliferation and cell cycle analysis were performed using flow cytometry. Apoptosis of PASMCs was determined using annexin V staining and cell migration was tested by wound healing assay. Results EZH2 protein expression in mouse PASMCs were correlated with an increase in right ventricular systolic pressure and Right Ventricular Hypertrophy (RVH). The overexpression of EZH2 in human PASMCs enhances proliferation, migration, and decrease in the rate of apoptosis when compared to GFP-transfected cells. In the G2/M phase of the EZH2 transfected cells, there was a 3.5 fold increase in proliferation, while there was a significant decrease in the rate of apoptosis of PASMCs, when compared to control. Conclusion These findings suggest that EZH2 plays a role in the migration and proliferation of PASMCs, which is a major hallmark in PAH. It also suggests that EZH2 could play a role in the development of PAH and can serve as a potential target for new therapies for PAH. PMID:22662197

Resveratrol Inhibits Proliferation, Invasion, and Epithelial-Mesenchymal Transition by Increasing miR-200c Expression in HCT-116 Colorectal Cancer Cells.

PubMed

Karimi Dermani, Fatemeh; Saidijam, Massoud; Amini, Razieh; Mahdavinezhad, Ali; Heydari, Korosh; Najafi, Rezvan

2017-06-01

colorectal cancer (CRC) is one of the most common malignancies, associated with high rates of relapse. A notable challenge in treatment is low response rate to current therapies for advanced CRC. The miR-200c plays an essential role in tumor suppression by inhibiting epithelial-mesenchymal transition (EMT). Resveratrol, a natural compound found in red wine, reveals anti-cancer properties in several types of cancers such as CRC. The aim of current study was to evaluate the effects of resveratrol on proliferation, apoptosis, and invasion of HCT-116 cells and also expression of EMT-related genes in presences or absence of miR-200c. the effect of resveratrol on viability was examined by MTT assay. LNA-anti-miR-200c transfection of HCT-116 cells was carried out in a time dependent manner. Then, the expression of miR-200c and EMT-related genes were quantified by qRT-PCR. Further, expression of EMT-related proteins, apoptosis, and invasion were analyzed by Western blot, Annexin V/PI staining and scratch test, respectively. resveratrol could significantly inhibit viability of HCT-116 cells. LNA-anti-miR-200c suppressed the endogenous miR-200c in transfected cells compared with the control. qRT-PCR and Western blot analysis of LNA-anti-miR-200c transfected cells revealed a considerable increase in vimentin and ZEB-1 expression, with a concomitant reduction in E-cadherin expression level. Migration of HCT-116 cells increased, and apoptosis significantly reduced in transfected cells. While, resveratrol could entirely reverse these changes by modulation of miR-200c expression. our findings revealed a major role of resveratrol in apoptosis, invasion, and switching of EMT to MET phenotype through upregulation of miR-200c in CRC. J. Cell. Biochem. 118: 1547-1555, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

PubMed Central

Do, Eun Kyoung; Cheon, Hyo Cheon; Heo, Soon Chul; Kwon, Yang Woo; Jeong, Geun Ok; Kim, Ba Reun; Kim, Jae Ho

2013-01-01

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate. PMID:24098810

Enhancing T cell activation and antiviral protection by introducing the HIV-1 protein transduction domain into a DNA vaccine.

PubMed

Leifert, J A; Lindencrona, J A; Charo, J; Whitton, J L

2001-10-10

Protein transduction domains (PTD), which can transport proteins or peptides across biological membranes, have been identified in several proteins of viral, invertebrate, and vertebrate origin. Here, we evaluate the immunological and biological consequences of including PTD in synthetic peptides and in DNA vaccines that contain CD8(+) T cell epitopes from lymphocytic choriomeningitis virus (LCMV). Synthetic PTD-peptides did not induce detectable CD8(+) T cell responses. However, fusion of an open reading frame encoding a PTD to an epitope minigene caused transfected tissue culture cells to stimulate epitope-specific T cells much more effectively. Kinetic studies indicated that the epitope reached the surface of transfected cells more rapidly and that the number of transfected cells needed to stimulate T cell responses was reduced by 35- to 50-fold when compared to cells transfected with a standard minigene plasmid. The mechanism underlying the effect of PTD linkage is not clear, but transit of the PTD-attached epitope from transfected cells to nontransfected cells (cross presentation) seemed to play, at most, a minimal role. Mice immunized once with the plasmid encoding the PTD-linked epitope showed a markedly accelerated CD8(+) T cell response and, unlike mice immunized with a standard plasmid, were completely protected against a normally lethal LCMV challenge administered only 8 days post-immunization.

LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

PubMed Central

Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

2009-01-01

Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

PubMed Central

Xia, Li; Yin, Shankai; Wang, Jian

2012-01-01

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

PubMed Central

Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean

2017-01-01

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamaguchi, Kazuki; Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180; Feril, Loreto B., E-mail: ferilism@yahoo.com

2011-07-22

Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA,more » which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.« less

Photoenhanced gene transfection by a curcumin loaded CS-g-PZLL micelle.

PubMed

Lin, Jian-Tao; Pan, Wen-Jia; Zhang, Jun-Ai; Wang, Wei; Zhong, Jia; Su, Jia-Min; Li, Tong; Zou, Ying; Wang, Guan-Hai

2017-09-01

The codelivery of drug and gene is a promising method for cancer treatment. In our previous works, we prepared a cationic micelles based on chitosan and poly-(N-3-carbobenzyloxylysine) (CS-g-PZLL), but transfection ratio of CS-g-PZLL to Hela cell was low. Herein, to improve the transfection efficiency of CS-g-PZLL, curcumin was loaded in the CS-g-PZLL micelle. After irradiation, the obtained curcumin loaded micelle showed a better transfection, and the p53 protein expression in Hela cells was higher. The apoptosis assay showed that the complex could induce a more significant apoptosis to Hela cells than that of curcumin or p53 used alone, and the curcumin loaded micelle inducing apoptosis was best after irradiation. Therefore, CS-g-PZLL is a safe and effective carrier for the codelivery of drug/gene, and curcumin could be used as a photosensitizer to induce a photoenhanced gene transfection, which should be encouraged in improving transfection and tumor therapy. Copyright © 2017. Published by Elsevier B.V.

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

PubMed

Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

2014-05-01

To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine. Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.

Development of polymeric-cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery.

PubMed

Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

2015-01-01

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid-polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

PubMed Central

Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

2015-01-01

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA. PMID:26648722

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

[Overexpression of SEPP1 inhibits the proliferation and induces cell cycle G2/M arrest of 786-O and 769-P human renal carcinoma cells].

PubMed

Liu, Kan; Zhao, Chaofei; Chen, Jianwen; Wu, Shengpan; Yao, Yuanxin; Wu, Chong; Luo, Guoxiong; Zhang, Xu

2016-06-01

Objective To establish selenoprotein P, plasma 1 (SEPP1) gene recombinant lentiviral vector and investigate the effect of SEPP1 on the proliferation of human clear cell renal cell carcinoma (ccRCC) cells. Methods cDNA sequence of SEPP1 was cloned from the total cDNA of HEK293T cells by PCR. Then, the cDNA fragment was combined with the pLV-EGFP(2A)Puro vector and the constructed plasmid pLV-EGFP(2A)Puro-SEPP1 was transfected into HEK293T cells for packaging the virus. Forty-eight hours after transfected with the virus supernatant, the level of SEPP1 protein in 769-P and 786-O cells were tested by Western blotting. Cells were divided into recombinant lentivirus-infected cells, empty vector lentivirus-infected cells and the blank control cells. Cell proliferation rate was detected by MTS assay, colony forming ability was evaluated by plate clony formation assay and cell cycle change was assayed by flow cytometry after transfected with pLV-EGFP(2A)Puro-SEPP1 or empty pLV-EGFP(2A)Puro vector. Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 was constructed successfully. After being infected by the virus supernatant, the 786-O and 769-P cells expressed EGFP. Compared with the empty vector group and the blank control group, expression level of SEPP1 in the experimental group was much higher. The cell proliferative ability was inhibited in the cells overexpressing SEPP1, and the colony forming ability of SEPP1-overexpressed cells evidently decreased. Cell cycle was arrested in G2/M phase in 786-O cells overexpressing SEPP1. Conclusion The recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 has been constructed successfully. Overexpression of SEPP1 could significantly reduce the proliferation rate of 786-O and 769P cells, and cause G2/M phase arrest of 786-O cells.

EGFP-EGF1-Conjugated PLGA Nanoparticles for Targeted Delivery of siRNA into Injured Brain Microvascular Endothelial Cells for Efficient RNA Interference

PubMed Central

Chen, Chen; Mei, Heng; Shi, Wei; Deng, Jun; Zhang, Bo; Guo, Tao; Wang, Huafang; Hu, Yu

2013-01-01

Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer. PMID:23593330

Cationic niosomes an effective gene carrier composed of novel spermine-derivative cationic lipids: effect of central core structures.

PubMed

Opanasopit, Praneet; Leksantikul, Lalita; Niyomtham, Nattisa; Rojanarata, Theerasak; Ngawhirunpat, Tanasait; Yingyongnarongkul, Boon-Ek

2017-05-01

Cationic niosomes formulated from Span 20, cholesterol (Chol) and novel spermine-based cationic lipids of multiple central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) were successfully prepared for improving transfection efficiency in vitro. The niosomes composed of spermine cationic lipid with central core structure of di(oxyethyl)amino revealed the highest gene transfection efficiency. To investigate the factors affecting gene transfection and cell viability including differences in the central core structures of cationic lipids, the composition of vesicles, molar ratio of cationic lipids in formulations and the weight ratio of niosomes to DNA. Cationic niosomes composed of nonionic surfactants (Span20), cholesterol and spermine-based cationic lipids of multiple central core structures were formulated. Gene transfection and cell viability were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge were also characterized. High transfection efficiency was obtained from cationic niosomes composed of Span20:Chol:cationic lipid at the molar ratio of 2.5:2.5:0.5 mM. Cationic lipids with di(oxyethyl)amino as a central core structure exhibited highest transfection efficiency. In addition, there was also no serum effect on transfection efficiency. These novel cationic niosomes may constitute a good alternative carrier for gene transfection.

Transient transfection of mammalian cells using a violet diode laser

NASA Astrophysics Data System (ADS)

Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

2010-07-01

We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

Effects of RNAi-mediated MUC4 gene silencing on the proliferation and migration of human pancreatic carcinoma BxPC-3 cells.

PubMed

Li, Yong; Wu, Changqiang; Chen, Tianwu; Zhang, Juanjuan; Liu, Gang; Pu, Yu; Zhu, Jiang; Shen, Chengyi; Zhang, Yang; Zeng, Nanlin; Zhang, Xiaoming

2016-12-01

It was previously demonstrated that mucin 4 (MUC4) is not expressed in normal pancreatic tissues or in chronic pancreatitis tissue but is highly expressed in pancreatic cancer (PC) tissue. Effective MUC4 gene knockdown in PC may contribute to the elucidation of pancreatic tumor development and metastasis, and may be valuable in new therapeutic approaches. Thus to confirm this, in the present study, the BxPC-3 cell line was transfected with eight pairs of shRNA lentiviral vectors for MUC4. The qPCR results showed that expression of MUC4 mRNA in the BxPC-3 cells was significantly decreased at 96 h after transfection. One of these shRNA lentiviral vectors (shRNA‑A141) had showed the strongest suppressive effect on MUC4 mRNA expression and was used for MUC4 knockdown in BxPC-3 cells. After stable transfection, BxPC-3 cells showed a significantly lower expression of MUC4 mRNA and MUC4 protein, and were suppressed on cell growth and migration. In vivo, lower tumor growth rates and tumor volume were observed in the tumors derived from the MUC4-knockdown cells, whereas the transplanted tumors derived from the control group cells, grew rapidly. Thus, inhibition of MUC4 expression may be an effective means for mitigating metastasis and invasion of PC.

From oleic acid-capped iron oxide nanoparticles to polyethyleneimine-coated single-particle magnetofectins

NASA Astrophysics Data System (ADS)

Cruz-Acuña, Melissa; Maldonado-Camargo, Lorena; Dobson, Jon; Rinaldi, Carlos

2016-09-01

Various inorganic nanoparticle designs have been developed and used as non-viral gene carriers. Magnetic gene carriers containing polyethyleneimine (PEI), a well-known transfection agent, have been shown to improve DNA transfection speed and efficiency in the presence of applied magnetic field gradients that promote particle-cell interactions. Here we report a method to prepare iron oxide nanoparticles conjugated with PEI that: preserves the narrow size distribution of the nanoparticles, conserves magnetic properties throughout the process, and results in efficient transfection. We demonstrate the ability of the particles to electrostatically bind with DNA and transfect human cervical cancer (HeLa) cells by the use of an oscillating magnet array. Their transfection efficiency is similar to that of Lipofectamine 2000™, a commercial transfection reagent. PEI-coated particles were subjected to acidification, and acidification in the presence of salts, before DNA binding. Results show that although these pre-treatments did not affect the ability of particles to bind DNA they did significantly enhanced transfection efficiency. Finally, we show that these magnetofectins (PEI-MNP/DNA) complexes have no effect on the viability of cells at the concentrations used in the study. The systematic preparation of magnetic vectors with uniform physical and magnetic properties is critical to progressing this non-viral transfection technology.

MicroRNA-133b targets glutathione S-transferase π expression to increase ovarian cancer cell sensitivity to chemotherapy drugs.

PubMed

Chen, Shuo; Jiao, Jin-Wen; Sun, Kai-Xuan; Zong, Zhi-Hong; Zhao, Yang

2015-01-01

Accumulating studies reveal that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. The aim of this study was to investigate the role of miR-133b in the development of drug resistance in ovarian cancer cells. We examined the levels of miR-133b expression in ovarian carcinoma tissues and the human ovarian carcinoma cell lines (A2780, A2780/DDP and A2780/Taxol, respectively). We determined the cell viability of these cell lines treated with cisplatin or paclitaxel in the presence or absence of miR-133b or anti-miR-133b transfection using the MTT assay. Reverse transcription polymerase chain reaction and Western blotting were used to assess the mRNA and protein expression levels of two drug-resistance-related genes: glutathione S-transferase (GST)-π and multidrug resistance protein 1 (MDR1). The dual-luciferase reporter assay was used to detect the promoter activity of GST-π in the presence and absence of miR-133b. The expression of miR-133b was significantly lower in primary resistant ovarian carcinomas than in the chemotherapy-sensitive carcinomas (P<0.05), and the same results were found in primary resistant ovarian cell lines (A2780/Taxol and A2780/DDP) compared to the chemotherapy-sensitive cell line (A2780; P<0.05). Following miR-133b transfection, four cell lines showed increased sensitivity to paclitaxel and cisplatin, while anti-miR-133b transfection reduced cell sensitivity to paclitaxel and cisplatin. Dual-luciferase reporter assay showed that miR-133b interacted with the 3'-untranslated region of GST-π. Compared to controls, the mRNA and protein levels of MDR1 and GST-π were downregulated after miR-133b transfection and upregulated after anti-miR-133b transfection. MicroRNA-133b may reduce ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GST-π and MDR1. In future, the combination of miR-133b with chemotherapy agents may prevent the development of drug resistance in ovarian cancers.

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Guinea pig adenovirus infection does not inhibit cochlear transfection with human adenoviral vectors in a model of hearing loss.

PubMed

Hankenson, F Claire; Wathen, Asheley B; Eaton, Kathryn A; Miyazawa, Toru; Swiderski, Donald L; Raphael, Yehoash

2010-04-01

Routine surveillance of guinea pigs maintained within a barrier facility detected guinea pig adenovirus (GPAdV) in sentinel animals. These guinea pigs served as models of induced hearing loss followed by regeneration of cochlear sensory (hair) cells through transdifferentiation of nonsensory cells by using human adenoviral (hAV) gene therapy. To determine whether natural GPAdV infection affected the ability of hAV vectors to transfect inner ear cells, adult male pigmented guinea pigs (n = 7) were enrolled in this study because of their prolonged exposure to GPAdV-seropositive conspecifics. Animals were deafened chemically (n = 2), received an hAV vector carrying the gene for green fluorescent protein (hAV-GFP) surgically without prior deafening (n = 2), or were deafened chemically with subsequent surgical inoculation of hAV-GFP (n = 3). Cochleae were evaluated by using fluorescence microscopy, and GFP expression in supporting cells indicated that the hAV-GFP vector was able to transfect inner ears in GPAdV-seropositive guinea pigs that had been chemically deafened. Animals had histologic evidence of interstitial pneumonia, attributable to prior infection with GPAdV. These findings confirmed that the described guinea pigs were less robust animal models with diminished utility for the overall studies. Serology tests confirmed that 5 of 7 animals (71%) were positive for antibodies against GPAdV at necropsy, approximately 7 mo after initial detection of sentinel infection. Control animals (n = 5) were confirmed to be seronegative for GPAdV with clinically normal pulmonary tissue. This study is the first to demonstrate that natural GPAdV infection does not negatively affect transfection with hAV vectors into guinea pig inner ear cells, despite the presence of other health complications attributed to the viral infection.

Protein Phosphatase 2A Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung

PubMed Central

Wallace, Alison M.; Hardigan, Andrew; Geraghty, Patrick; Salim, Shaneeza; Gaffney, Adam; Thankachen, Jincy; Arellanos, Leo; D'Armiento, Jeanine M.; Foronjy, Robert F.

2012-01-01

Protein phosphatase 2A (PP2A) is the primary serine-threonine phosphatase of eukaryotic cells, and changes in its activity have been linked to neoplastic and neurodegenerative diseases. However, the role of PP2A in noncancerous lung diseases such as chronic obstructive pulmonary disease (COPD) has not been previously examined. This study determined that PP2A activity was significantly increased in the lungs of advanced emphysema subjects compared with age-matched controls. Furthermore, we found that cigarette smoke exposure increases PP2A activity in mouse lung in vivo and in primary human small airway epithelial (SAE) cells in vitro. In mice, intratracheal transfection of PP2A protein prior to cigarette smoke exposure prevented acute smoke–induced lung inflammation. Conversely, inhibiting PP2A activity during smoke exposure exacerbated inflammatory responses in the lung. To further determine how PP2A modulates the responses to cigarette smoke in the lung, enzyme levels were manipulated in SAE cells using protein transfection and short hairpin RNA (shRNA) techniques. Increasing PP2A activity in SAE cells via PP2A protein transfection downregulated cytokine expression and prevented the induction of proteases following cigarette smoke extract (CSE) treatment. Conversely, decreasing enzymatic activity by stably transfecting SAE cells with shRNA for the A subunit of PP2A exacerbated these smoke-mediated responses. This study establishes that PP2A induction by cigarette smoke modulates immune and proteolytic responses to cigarette smoke exposure. Together, these findings suggest that manipulation of PP2A activity may be a plausible means to treat COPD and other inflammatory diseases. PMID:22223484

CADASIL mutant NOTCH3(R90C) decreases the viability of HS683 oligodendrocytes via apoptosis.

PubMed

Tang, Mibo; Shi, Changhe; Song, Bo; Yang, Jing; Yang, Ting; Mao, Chengyuan; Li, Yusheng; Liu, Xinjing; Zhang, Shuyu; Wang, Hui; Luo, Haiyang; Xu, Yuming

2017-07-01

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary cerebral small vessel disease caused by mutations in NOTCH3. Prevailing models suggest that demyelination occurs secondary to vascular pathology. However, in zebrafish, NOTCH3 is also expressed in mature oligodendrocytes. Thus, we hypothesized that in addition to vascular defects, mutant NOTCH3 may alter glial function in individuals with CADASIL. The aim of this study was to characterize the direct effects of a mutant NOTCH3 protein in HS683 oligodendrocytes. HS683 oligodendrocytes transfected with wild-type NOTCH3, mutant NOTCH3(R90C), and empty control vector were used to study the impact of the NOTCH3(R90C) mutant on its protein hydrolytic processing, cell viability, apoptosis, autophagy, oxidative stress, and the related upstream events using immunoblotting, immunofluorescence, RT-PCR, and flow cytometry. We determined that HS683 oligodendrocytes transfected with mutant NOTCH3(R90C), which is the hotspot mutation site-associated with CADASIL, exhibited aberrant NOTCH3 proteolytic processing. Compared to cells overexpressing wild-type NOTCH3, cells overexpressing NOTCH3(R90C) were less viable and had a higher rate of apoptosis. Immunoblotting revealed that cells transfected with NOTCH3(R90C) had higher levels of intrinsic mitochondrial apoptosis, extrinsic death receptor path-related apoptosis, and autophagy compared with cells transfected with wild-type NOTCH3. This study suggests that in patients with CADASIL, early defects in glia influenced by NOTCH3(R90C) may directly contribute to white matter pathology in addition to secondary vascular defects. This study provides a potential therapeutic target for the future treatment of CADASIL.

Chitosan nanoparticle carrying small interfering RNA to platelet-derived growth factor B mRNA inhibits proliferation of smooth muscle cells in rabbit injured arteries.

PubMed

Xia, He; Jun, Ji; Wen-Ping, Ling; Yi-Feng, Pan; Xiao-Ling, Chen

2013-10-01

The purpose of this study was to elucidate the transfection of chitosan nanoparticle carrying small interfering RNA against platelet-derived growth factor B (PDGF-B) to inhibit the expression of PDGF-B mRNA and proliferation of smooth muscle cells. A rabbit iliac artery injury model was constructed. A small interfering RNA (siRNA) against PDGF-B mRNA expression vector was constructed and packaged by chitosan nanoparticle to transfect into the vascular smooth muscle cells (vSMCs) of balloon catheter-injured rabbit iliac artery wall, using a therapeutic ultrasound for the gene delivery. The experiment was divided into two groups: experimental group, denudation and nano-PDGF-B siRNA treated, and only single denudation as control. Effects of the siRNA on the expressions of proliferating cell nuclear antigen (PCNA) and PDGF-B mRNA by vSMCs and the proliferation of vSMCs were observed with the methods of routine pathological, immunohistochemical staining, in situ hybridization and morphometry. The nano siRNA against PDGF-B was successfully transfected. The nano siRNA significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs. The local intimal thickness and area were also reduced remarkably. In conclusion, transfection of chitosan nanoparticle carrying siRNA against PDGF-B mRNA could inhibit proliferation of vSMCs in the rabbit iliac artery injury model. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Neurotrophins differentially stimulate the growth of cochlear neurites on collagen surfaces and in gels☆

PubMed Central

Xie, Joanna; Pak, Kwang; Evans, Amaretta; Kamgar-Parsi, Andy; Fausti, Stephen; Mullen, Lina; Ryan, Allen Frederic

2013-01-01

The electrodes of a cochlear implant are located far from the surviving neurons of the spiral ganglion, which results in decreased precision of neural activation compared to the normal ear. If the neurons could be induced to extend neurites toward the implant, it might be possible to stimulate more discrete subpopulations of neurons, and to increase the resolution of the device. However, a major barrier to neurite growth toward a cochlear implant is the fluid filling the scala tympani, which separates the neurons from the electrodes. The goal of this study was to evaluate the growth of cochlear neurites in three-dimensional extracellular matrix molecule gels, and to increase biocompatibility by using fibroblasts stably transfected to produce neurotrophin-3 and brain-derived neurotrophic factor. Spiral ganglion explants from neonatal rats were evaluated in cultures. They were exposed to soluble neurotrophins, cells transfected to secrete neurotrophins, and/or collagen gels. We found that cochlear neurites grew readily on collagen surfaces and in three-dimensional collagen gels. Co-culture with cells producing neurotrophin-3 resulted in increased numbers of neurites, and neurites that were longer than when explants were cultured with control fibroblasts stably transfected with green fluorescent protein. Brain-derived neurotrophic factor-producing cells resulted in a more dramatic increase in the number of neurites, but there was no significant effect on neurite length. It is suggested that extracellular matrix molecule gels and cells transfected to produce neurotrophins offer an opportunity to attract spiral ganglion neurites toward a cochlear implant. PMID:24459465

MicroRNA-873 mediates multidrug resistance in ovarian cancer cells by targeting ABCB1.

PubMed

Wu, Di-di; Li, Xue-Song; Meng, Xiao-Na; Yan, Jing; Zong, Zhi-Hong

2016-08-01

Ovarian cancer is commonly treated with cisplatin and paclitaxel combination chemotherapy; however, ovarian cancer cells often develop resistance to these drugs. Increasingly, microRNAs (miRNAs) including miR-873 have been implicated in drug resistance in many cancers, but the role of miR-873 in ovarian cancer remains unknown. MTT cell viability assays revealed that the sensitivities of ovarian cancer lines to cisplatin and paclitaxel increased following transfection with miR-873 (P < 0.05). After predicting the miR-873 binding region in the 3'-untranslated region of ABCB1, dual-luciferase reporter assay confirmed this prediction. RT-PCR and Western blotting revealed that MDR1 expression was significantly downregulated after transfection with miR-873 and upregulated after transfection with anti-miR-873 at both mRNA and protein levels compared to negative controls (P < 0.05). Experiments in a mouse xenograft model confirmed that intratumoral administration of miR-873 could enhance the efficacy of cisplatin in inhibiting tumor growth in ovarian cancer in vivo (P < 0.05). ABCB1 overexpression reduced sensitivities of ovarian cancer lines OVCAR3 and A2780 to cisplatin and paclitaxel, which can be reversed by miR-873 mimic transfection (P < 0.05). In summary, we demonstrated that overexpression of miR-873 increased the sensitivity of ovarian cancer cells to cisplatin and paclitaxel by targeting MDR1 expression. Our findings suggest that combination therapies with chemotherapy agents and miR-873 may suppress drug resistance in ovarian cancer.

Introduction of OX40 ligand into lymphoma cells elicits anti-lymphoma immunity in vivo.

PubMed

Kaneko, Hitomi; Hori, Toshiyuki; Yanagita, Soshi; Kadowaki, Norimitsu; Uchiyama, Takashi

2005-03-01

OX40, a member of the TNF receptor superfamily, and its ligand (OX40L) play crucial roles in induction and maintenance of integrated T cell immune response. Engagement of OX40L delivers a costimulatory signal to T cells. In this study, we investigated whether inoculation of OX40L-transfected EL4, a murine T cell lymphoma cell line, could induce anti-lymphoma immunity in mice. Female C57BL/6 mice were inoculated with 1 x 10(5) cells of parental EL4, OX40L-transfected EL4 (EL4-OX40L), or mock control vector-transfected EL4 (EL4-mock), and then the tumor size, overall survival, CTL activity of spleen cells, and the immunohistochemistry were compared. While both parental EL4 and EL4-mock grew rapidly, EL4-OX40L was rejected or grew slower than parental EL4 or EL4-mock. Pretreatment of mice with either anti-CD4 or anti-CD8 mAb accelerated the growth of EL4-OX40L, suggesting that both CD4+ and CD8+ T cells were involved in anti-lymphoma immunity. The immunohistochemical study revealed the infiltration of CD8+ T cells into the tumor of EL4-OX40L. In vitro CTL assay demonstrated that spleen cells of mice that had rejected EL4-OX40L had significant cytotoxic activity against parental EL4. The gene transfer of OX40L into lymphoma cells is an eligible and efficient modality to induce anti-lymphoma immunity.

Optical transfection using an endoscope-like system.

PubMed

Ma, Nan; Gunn-Moore, Frank; Dholakia, Kishan

2011-02-01

Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ∼ 6000 cores (pixels) embedded in a fiber cladding of ∼ 300 μm in diameter, produces an image circle (area) of ∼ 270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ∼ 72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

PubMed

Eshima, Koji; Misawa, Kana; Ohashi, Chihiro; Iwabuchi, Kazuya

2018-05-01

Although CD4 + T cells are generally regarded as helper T cells, some activated CD4 + T cells have cytotoxic properties. Given that CD4 + cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4 + T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4 + T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4 + T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4 + T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4 + T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas - target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Japanese encephalitis virus non-coding RNA inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3.

PubMed

Chang, Ruey-Yi; Hsu, Ta-Wen; Chen, Yen-Lin; Liu, Shu-Fan; Tsai, Yi-Jer; Lin, Yun-Tong; Chen, Yi-Shiuan; Fan, Yi-Hsin

2013-09-27

Noncoding RNA (ncRNA) plays a critical role in modulating a broad range of diseases. All arthropod-borne flaviviruses produce short fragment ncRNA (sfRNA) collinear with highly conserved regions of the 3'-untranslated region (UTR) in the viral genome. We show that the molar ratio of sfRNA to genomic RNA in Japanese encephalitis virus (JEV) persistently infected cells is greater than that in acutely infected cells, indicating an sfRNA role in establishing persistent infection. Transfecting excess quantities of sfRNA into JEV-infected cells reduced interferon-β (IFN-β) promoter activity by 57% and IFN-β mRNA levels by 52%, compared to mock-transfected cells. Transfection of sfRNA into JEV-infected cells also reduced phosphorylation of interferon regulatory factor-3 (IRF-3), the IFN-β upstream regulator, and blocked roughly 30% of IRF-3 nuclear localization. Furthermore, JEV-infected sfRNA transfected cells produced 23% less IFN-β-stimulated apoptosis than mock-transfected groups did. Taken together, these results suggest that sfRNA plays a role against host-cell antiviral responses, prevents cells from undergoing apoptosis, and thus contributes to viral persistence. Copyright © 2013 Elsevier B.V. All rights reserved.

Transfection of apoptosis related gene Fas ligand in human hepatocellular carcinoma cells and its significance in apoptosis

PubMed Central

Chen, Jun; Su, Xian-Shi; Jiang, Yong-Fang; Gong, Guo-Zhong; Zheng, Yu-Huang; Li, Gui-Yuan

2005-01-01

AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immuohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope. RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining. CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells. PMID:15849828

The effect of environmental pH on polymeric transfection efficiency.

PubMed

Kang, Han Chang; Samsonova, Olga; Kang, Sun-Woong; Bae, You Han

2012-02-01

Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6-7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1-2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. Copyright © 2011 Elsevier Ltd. All rights reserved.

MicroRNA-15b regulates reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression in human uterine leiomyoma.

PubMed

Guan, Yichun; Guo, Lankai; Zukerberg, Lawrence; Rueda, Bo R; Styer, Aaron K

2016-08-17

Human uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied. The expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor. Elevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p < 0.0001) and pLYO cells (0.68-fold; p < 0.0001) demonstrated reduced RECK protein expression. Following transfection with miR-15b inhibitor, cpLYO cells (1.20-fold; p < 0.0001) and pLYO cells (1.31-fold; p = 0.0007) demonstrated elevated RECK protein expression. RECK protein expression was reduced in pLYO tissues (0.73-fold; p < 0.0001) and pLYO (0.47-fold; p = 0.047) cells when compared to the corresponding MYO tissue controls. Our findings suggest that miR-15b negatively regulates RECK expression in LYO, and increased miR-15b and decreased RECK expression may contribute to the pathobiology of LYO. The functional significance of miR-15b and RECK expression warrants further investigation as potential therapeutic targets for the treatment of human LYO.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

PubMed

Chernousova, S; Epple, M

2017-05-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent

PubMed Central

Chernousova, S; Epple, M

2017-01-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2–3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application. PMID:28218744

The use of biodegradable PLGA nanoparticles to mediate SOX9 gene delivery in human mesenchymal stem cells (hMSCs) and induce chondrogenesis.

PubMed

Kim, Jae-Hwan; Park, Ji Sun; Yang, Han Na; Woo, Dae Gyun; Jeon, Su Yeon; Do, Hyun-Jin; Lim, Hye-Young; Kim, Jung Mo; Park, Keun-Hong

2011-01-01

In stem cell therapy, transfection of specific genes into stem cells is an important technique to induce cell differentiation. To perform gene transfection in human mesenchymal stem cells (hMSCs), we designed and fabricated a non-viral vector system for specific stem cell differentiation. Several kinds of gene carriers were evaluated with regard to their transfection efficiency and their ability to enhance hMSCs differentiation. Of these delivery vehicles, biodegradable poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles yielded the best results, as they complexed with high levels of plasmid DNA (pDNA), allowed robust gene expression in hMSCs, and induced chondrogenesis. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. The expression of enhanced green fluorescent protein (EGFP) and SOX9 increased up to 75% in hMSCs transfected with PEI/SOX9 complexed PLGA nanoparticles 2 days after transfection. SOX9 gene expression was evaluated by RT-PCR, real time-qPCR, glycosaminoglycan (GAG)/DNA levels, immunoblotting, histology, and immunofluorescence. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effect of NET-1 siRNA conjugated sub-micron bubble complex combined with low-frequency ultrasound exposure in gene transfection

PubMed Central

Wu, Bolin; Liang, Xitian; Jing, Hui; Han, Xue; Sun, Yixin; Guo, Cunli; Liu, Ying; Cheng, Wen

2018-01-01

The present study evaluated the effect of NET-1 siRNA-conjugated sub-micron bubble (SMB) complexes combined with low-frequency ultrasound exposure in gene transfection. The NET-1 gene was highly expressed level in SMMC-7721 human hepatocellular carcinoma cell line. The cells were divided into seven groups and treated with different conditions. The groups with or without low-frequency ultrasound exposure, groups of adherent cells, and suspension cells were separated. The NET-1 siRNA-conjugated SMB complexes were made in the laboratory and tested by Zetasizer Nano ZS90 analyzer. Flow cytometry was used to estimate the transfection efficiency and cellular apoptosis. Western blot and quantitative real-time polymerase chain reaction (qPCR) were used for the estimation of the protein and mRNA expressions, respectively. Transwell analysis determined the migration and invasion capacities of the tumor cells. The results did not show any difference in the transfection efficiency between adherent and suspension cells. However, the NET-1 siRNA-SMB complexes combined with low-frequency ultrasound exposure could enhance the gene transfection effectively. In summary, the NET-1 siRNA-SMB complexes appeared to be promising gene vehicle. PMID:29423111

Controlling DNA compaction with cationic amphiphiles for efficient delivery systems A step forward towards non-viral Gene Therapy

NASA Astrophysics Data System (ADS)

Savarala, Sushma

The synthesis of pyridinium cationic lipids, their counter-ion exchange, and the transfection of lipoplexes consisting of these lipids with firefly luciferase plasmid DNA (6.7 KDa), into lung, prostate and breast cancer cell lines was investigated. The transfection ability of these newly synthesized compounds was found to be twice as high as DOTAP/cholesterol and Lipofectamine TM (two commercially available successful transfection agents). The compaction of the DNA onto silica (SiO2) nanoparticles was also investigated. For this purpose, it was necessary to study the stability and fusion studies of colloidal systems composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), a zwitterionic lipid, and mixtures of DMPC with cationic DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane).

All optical experimental design for neuron excitation, inhibition, and action potential detection

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Tolstykh, Gleb; Martens, Stacey; Sedelnikova, Anna; Ibey, Bennett L.; Beier, Hope T.

2016-03-01

Recently, infrared light has been shown to both stimulate and inhibit excitatory cells. However, studies of infrared light for excitatory cell inhibition have been constrained by the use of invasive and cumbersome electrodes for cell excitation and action potential recording. Here, we present an all optical experimental design for neuronal excitation, inhibition, and action potential detection. Primary rat neurons were transfected with plasmids containing the light sensitive ion channel CheRiff. CheRiff has a peak excitation around 450 nm, allowing excitation of transfected neurons with pulsed blue light. Additionally, primary neurons were transfected with QuasAr2, a fast and sensitive fluorescent voltage indicator. QuasAr2 is excited with yellow or red light and therefore does not spectrally overlap CheRiff, enabling imaging and action potential activation, simultaneously. Using an optic fiber, neurons were exposed to blue light sequentially to generate controlled action potentials. A second optic fiber delivered a single pulse of 1869nm light to the neuron causing inhibition of the evoked action potentials (by the blue light). When used in concert, these optical techniques enable electrode free neuron excitation, inhibition, and action potential recording, allowing research into neuronal behaviors with high spatial fidelity.

Characterization of insulin-producing cells derived from PDX-1-transfected neural stem cells.

PubMed

Wang, Hailan; Jiang, Zesheng; Li, Aihui; Gao, Yi

2012-12-01

Islet cell transplantation is a promising treatment strategy for type-1 diabetes. However, functional islet cells are hard to obtain for transplantation and are in short supply. Directing the differentiation of stem cells into insulin‑producing cells, which serve as islet cells, would overcome this shortage. Bone marrow contains hematopoietic stem cells and mesenchymal stem cells. The present study used bone marrow cells isolated from rats and neural stem cells (NSCs) that were derived from bone marrow cells in culture. Strong nestin staining was detected in NSCs, but not in bone marrow stromal cells (BMSCs). In vitro transfection of the pancreatic duodenal homeobox-1 (PDX-1) gene into NSCs generated insulin‑producing cells. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed that PDX-1-transfected NSCs expressed insulin mRNA and released insulin protein. However, insulin release from PDX-1-transfected NSCs did not respond to the challenge of glucose and glucagon-like peptide-1. These results support the use of bone marrow-derived NSCs as a renewable source of insulin-producing cells for autologous transplantation to treat type-1 diabetes.

Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

NASA Technical Reports Server (NTRS)

Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

1998-01-01

The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

Three-dimensional imaging of nucleolin trafficking in normal cells, transfectants, and heterokaryons

NASA Astrophysics Data System (ADS)

Ballou, Byron T.; Fisher, Gregory W.; Deng, Jau-Shyong; Hakala, Thomas R.; Srivastava, Meera; Farkas, Daniel L.

1996-04-01

The study of intracellular trafficking using labeled molecules has been aided by the development of the cyanine fluorochromes, which are easily coupled, very soluble, resist photobleaching, and fluoresce at far-red wavelengths where background fluorescence is minimal. We have used Cy3-, Cy5-, and Cy5.5-labeled antibodies, antigen-binding fragments, and specifically binding single-stranded oligonucleotides to follow expression and trafficking of nucleolin, the most abundant protein of the nucleolus. Nucleolin shuttles between the nucleolus and the cytoplasm, and is also expressed on the cell surface, allowing us to test our techniques at all three cellular sites. Differentially cyanine-labeled non-specific antibodies were used to control for non-specific binding. Similarly, the differentially labeled non-binding strand of the cloned oligonucleotide served as a control. The multimode microscope allowed us to follow both rapid and slow redistributions of labeled ligands in the same study. We also performed 3-D reconstructions of nucleolin distribution in cells using rapid acquisition and deconvolution. Microinjection of labeled ligands was used to follow intracellular distribution, while incubation of whole cells with antibody and antigen-binding fragments was used to study uptake. To unambiguously define trafficking, and eliminate the possibility of interference by cross-reactive proteins, we transfected mouse renal cell carcinoma cells that express cell surface nucleolin with human nucleolin. We used microinjection and cell surface staining with Cy3- or Cy5- labeled monoclonal antibody D3 (specific for human nucleolin) to assess the cellular distribution of the human protein. Several clones expressed human nucleolin on their surfaces and showed high levels of transport of the human protein into the mouse nucleus and nucleolus. This distribution roughly parallels that of mouse nucleolin as determined by labeled polyclonal antibody. We have used these engineered transfectants to determine whether the cell surface-expressed xenogeneic nucleolin can serve as a target for antibodies in vivo.

Enhancement of Poly(orthoester) Microspheres for DNA Vaccine Delivery by Blending with Poly(ethylenimine)

PubMed Central

Nguyen, David N.; Raghavan, Shyam S.; Tashima, Lauren M.; Lin, Elizabeth C.; Fredette, Stephen J.; Langer, Robert S.; Wang, Chun

2008-01-01

Poly(ortho ester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced the efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy with labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE-PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50% to 60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE-PEI blended microspheres may be excellent carriers for DNA vaccines. PMID:18400294

[Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells].

PubMed

Chen, Dengyu; Chu, Yifan; Zheng, Qingwei; Xu, Zhiben; Zhou, Ping; Li, Sheng

2017-08-01

Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by Lipofectamine TM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by Transwell TM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.

Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

PubMed

Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

2007-02-20

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 microm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers

PubMed Central

Coulthard, Sally A.; Redfern, Christopher P.F.; Vikingsson, Svante; Appell, Malin Lindqvist; Skoglund, Karin; Falk, Ingrid Jakobsen; Hall, Andrew G.; Taylor, Gordon A.; Hogarth, Linda A.

2011-01-01

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP−ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP−ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP. PMID:21282358

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki

2014-05-15

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether directmore » cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.« less

H. pylori modifies methylation of global genomic DNA and the gastrin gene promoter in gastric mucosal cells and gastric cancer cells.

PubMed

Xie, Yuan; Zhou, Jian Jiang; Zhao, Yan; Zhang, Ting; Mei, Liu Zheng

2017-07-01

The aim of this study was to evaluate the correlation between H. pylori infection and global DNA methylation, as well as the methylation levels of the gastrin promoters. We constructed a eukaryotic expression vector, pcDNA3.1::cagA, and transfected it into GES-1 gastric mucosal cells and SGC-7901 gastric cancer cells. Both cell lines were infected with the H. pylori/CagA + strain NCTC11637. Then, we detected global DNA methylation by capture and detection antibodies, followed by colorimetric quantification. The methylation levels of the gastrin promoter were evaluated by base-specific cleavage and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In H. pylori/CagA + -infected GES-1 and SGC-7901 cells, the methylation levels of genomic DNA decreased by 49.4% and 18.8%, and in GES-1 and SGC-7901 cells transfected with pcDNA3.1::cagA, the methylation levels of genomic DNA decreased by 17.05% and 25.6%, respectively. Among 24 methylation sites detected in the gastrin promoter region, the methylation levels of 9 CpG sites were significantly decreased in H. pylori/CagA+-infected and pcDNA3.1:: cagA-transfected cells in comparison to corresponding control cells. These results indicate that H. pylori/CagA + decreases the methylation of the genome and the gastrin promoter at some CpG sites in gastric mucosal and gastric cancer cells. Copyright © 2017. Published by Elsevier Ltd.

Tumor suppression function of the Big-h3 gene in radiation carcinogenesis

NASA Astrophysics Data System (ADS)

Zhao, Y.; Piao, C.; Hei, T.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we show here that expression of Big-h3 gene, a secreted adhesion molecule induced by transforming growth factor- beta (TGF-beta ), is markedly decreased in independently generated, high LET radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Expression of this gene was restored to control level in fusion cell lines between the tumorigenic and parental BEP2D cells that were no longer tumorigenic in nude mice. Transfection of Big-h3 gene into tumor cells resulted in a significant reduction of tumor growth. While integrin receptor alpha 5/beta 1 was overexpressed in tumor cells, its expression was corrected to the level of control BEP2D cells after Big-h3 transfection. These data suggest that Big-h3 is involved in tumor progression by regulating integrin receptor alpha 5/beta 1. . WWee We further show that down regulation of Big-h3 results from loss of expression of TGFbeta1 in tumor cells. The findings provide strong evidence that the Big-h3 gene has tumor suppressor function in radiation induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.

Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle.

PubMed

Wongsrikeao, Pimprapar; Nagai, Takashi; Agung, Budiyanto; Taniguchi, Masayasu; Kunishi, Miho; Suto, Shizuyo; Otoi, Takeshige

2007-06-01

The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos. (c) 2006 Wiley-Liss, Inc.

Postmitotic Expression of SOD1G93A Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation

PubMed Central

Martini, Martina; Dobrowolny, Gabriella; Aucello, Michela; Musarò, Antonio

2015-01-01

To determine the role of mutant SOD1 gene (SOD1G93A) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1G93A gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1G93A in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1G93A gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1G93A gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1G93A gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1G93A in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1G93A on myogenic program and disclosed potential signaling, activated by SOD1G93A, that affect the identity of the myogenic cell population. PMID:26491230

Postmitotic Expression of SOD1(G93A) Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation.

PubMed

Martini, Martina; Dobrowolny, Gabriella; Aucello, Michela; Musarò, Antonio

2015-01-01

To determine the role of mutant SOD1 gene (SOD1(G93A)) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1(G93A) gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1(G93A) in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1(G93A) gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1(G93A) gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1(G93A) gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1(G93A) in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1(G93A) on myogenic program and disclosed potential signaling, activated by SOD1(G93A), that affect the identity of the myogenic cell population.

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

PubMed

Pastor, Marie; Johnen, Sandra; Harmening, Nina; Quiviger, Mickäel; Pailloux, Julie; Kropp, Martina; Walter, Peter; Ivics, Zoltán; Izsvák, Zsuzsanna; Thumann, Gabriele; Scherman, Daniel; Marie, Corinne

2018-06-01

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

[Impact of siRNA-mediated down-regulation of CD147 on human breast cancer cells].

PubMed

Li, Zhenqian; Li, Daoming; Li, Jiangwei; Huang, Pei; Qin, Hui

2015-10-01

To investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231. The protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC. Lentiviral expression vector of CD147 gene was constructed and transfected into MDA-MB-231 cells. RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD147 genes to identify the optimal time point, followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells. The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD147 siRNA on the tumor transplants. After transfection of lentiviral expression vector of CD147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed, significantly weaker than those of control group (P < 0.01). After 72 hours of transfection, average down-regulation rate of CD147 and MMP-2 were 96.03% ± 0.84% and 96.03% ± 0.84%, respectively. Both CD147 mRNA and MMP-2 mRNA expression were down-regulated (P < 0.05), while TIMP-2 mRNA expression showed no significant deference (P > 0.05). No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited (P < 0.05). After 24 hours of transection, average migration distance of MDA-MB-231 cell line and control group were (0.64 ± 0.12) mm and (4.69 ± 0.85) mm, respectively, which indicated a lower migrate speed. Down regulation of CD147 led to reduction of volume and mass of nude mouses. The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 (P < 0.05), with an average tumor mass of (1.85 ± 0.98) g and both reduction of tumor size and tumor mass. CD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice.

Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models

PubMed Central

Langdon, Jacqueline M.; Schroeder, John T.; Vonakis, Becky M.; Bieneman, Anja P.; Chichester, Kristin; MacDonald, Susan M.

2008-01-01

Previously, we demonstrated a negative correlation between histamine release to histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP-1 in human basophils. The present study was conducted to investigate whether suppressing SHIP-1 using small interfering (si)RNA technology would alter the releasability of culture-derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro-34 medium containing cytokines: mast cells with IL-6 and stem cell factor (100 ng/ml each) for 6–8 weeks and basophils with IL-3 (6.7 ng/ml) for 2–3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP-1 or a negative control siRNA. Changes in SHIP-1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP-induced histamine release. siRNA knockdown of SHIP-1 in mast cells ranged from 31% to 82%, mean 65 ± 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP-1 knockdown in basophils ranged from 34% to 69%, mean 51.8 ± 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP-1 protein in cultured mast cells and in cultured basophils increases releasability of the cells. PMID:18625911

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Li-An; Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an; Yuan, Guohua

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show thatmore » transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.« less

Modified model of VX2 tumor overexpressing vascular endothelial growth factor.

PubMed

Pascale, Florentina; Ghegediban, Saida-Homayra; Bonneau, Michel; Bedouet, Laurent; Namur, Julien; Verret, Valentin; Schwartz-Cornil, Isabelle; Wassef, Michel; Laurent, Alexandre

2012-06-01

To determine whether upregulated expression of vascular endothelial growth factor (VEGF) in VX2 cells can increase vessel density (VD) and reduce tumor necrosis. The VX2 cell line was transfected with expression vectors containing cDNA for rabbit VEGF. Stable clones producing rabbit VEGF (VEGF-VX2) were selected. VEGF-VX2 cells (n = 5 rabbits) or nontransfected VX2 cells (controls; n = 5 rabbits) were implanted into leg muscle of 10 rabbits. The animals were sacrificed at day 21. Tumor volume, percentage of necrosis, VD, and VEGF concentration in tumor protein extract were quantified. Overexpression of VEGF by VX2 cells augmented tumor implantation efficiency 100% and favored cyst formation. The tumor volume was significantly larger for VEGF-VX2 transfected tumors versus controls (P = .0143). Overexpression of VEGF in VX2 cells significantly increased the VD of the tumors (P = .0138). The percentage of necrosis was reduced in VEGF-VX2 tumors versus controls (19.5% vs 38.5 %; P = .002). VEGF concentration in VEGF-VX2 tumors was significantly higher than in control tumors (P = .041) and was correlated with tumor volume (ρ = .883, P = .012). The overexpression of VEGF increased tumor growth and vascularization, favored cyst formation, and reduced tumor necrosis. This new phenotype of the VX2 tumor may offer some advantages over classic models of VX2 tumor for evaluating anticancer therapies. Copyright © 2012 SIR. Published by Elsevier Inc. All rights reserved.

Astrocyte-specific expression of human T-cell lymphotropic virus type 1 (HTLV-1) Tax: induction of tumor necrosis factor alpha and susceptibility to lysis by CD8+ HTLV-1-specific cytotoxic T cells.

PubMed

Méndez, E; Kawanishi, T; Clemens, K; Siomi, H; Soldan, S S; Calabresi, P; Brady, J; Jacobson, S

1997-12-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with a chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP). Although the pathogenesis of this disease remains to be elucidated, the evidence suggests that immunopathological mechanisms are involved. Since HTLV-1 tax mRNA was colocalized with glial acidic fibrillary protein, a marker for astrocytes, we developed an in vitro model to assess whether HTLV-1 infection activates astrocytes to secrete cytokines or present viral immunodominant epitopes to virus-specific T cells. Two human astrocytic glioma cell lines, U251 and U373, were transfected with the 3' portion of the HTLV-1 genome and with the HTLV-1 tax gene under astrocyte-specific promoter control. In this study, we report that Tax-expressing astrocytic glioma transfectants activate the expression of tumor necrosis factor alpha mRNA in vitro. Furthermore, these Tax-expressing glioma transfectants can serve as immunological targets for HTLV-1-specific cytotoxic T lymphocytes (CTL). We propose that these events could contribute to the neuropathology of HAM/TSP, since infected astrocytes can become a source for inflammatory cytokines upon HTLV-1 infection and serve as targets for HTLV-1-specific CTL, resulting in parenchymal damage by direct lysis and/or cytokine release.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

PubMed

Weil, D; Garçon, L; Harper, M; Duménil, D; Dautry, F; Kress, M

2002-12-01

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

Cell Cycle Dependent Regulation of Human Progesterone Receptor in Breast Cancer

DTIC Science & Technology

2004-10-01

wt PR or S400A PR (0.01-1.0 [tg each), CDK2 (ljig), and PRE-2x-TATA-luc reporter plasmid (lig) along with Renilla plasmid (10ng) as a control for...8 hours prior to treatment. Cells were collected following treatment with 10 nM R5020 or ETOH vehicle control for 18 hrs. Luciferase and renilla ...reporter, a renilla reporter construct as a transfection control, either wt PR-B or S400A mutant PR-B, and control parental vector or a vector encoding an

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

PubMed Central

Snapp, Karen R.; Craig, Ron; Herron, Michael; Nelson, Robert D.; Stoolman, Lloyd M.; Kansas, Geoffrey S.

1998-01-01

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin. PMID:9660879

[Histocompatibility of nano-hydroxyapatite/poly-co-glycolic acid tissue engineering bone modified by mesenchymal stem cells with vascular endothelial frowth factor].

PubMed

Zhang, Minglei; Wang, Dapeng; Yin, Ruofeng

2015-10-06

To explorec Histocompatibility of nano-hydroxyapatite/poly-co-glycolic acid tissue engineering bone modified by mesenchymal stem cells with vascular endothelial frowth factor transinfected. Rat bone marrow mesenchymal stem cells (BMSCs) was separated, using BMSCs as target cells, and then vascular endothelial growth factor (VEGF) gene was transfected. Composite bone marrow mesenchymal stem cells and cells transfected with nano-hydroxyapatite (HA)/polylactic-co-glycolic acid (PLGA). The composition of cell and scaffold was observed. The blank plasmid transfection was 39.1%, 40.1% in VEGF group. The cell adhesion and growth was found on the scaffold pore wall after 5 days, and the number of adherent cells in the nano-HA/PLGA composite scaffold material basically had no significant difference in both. Although the nano-HA/PLGA scaffold material is still not fully meet the requirements of the matrix material for bone tissue engineering, but good biocompatibility, structure is its rich microporous satisfaction in material mechanics, toughening, enhanced obviously. Composition scaffold with BMSCs transfected by VEGF plasmid, the ability of angiogenesis is promoted.

Expression and analysis of exogenous proteins in epidermal cells.

PubMed

Dagnino, Lina; Ho, Ernest; Chang, Wing Y

2010-01-01

In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

[Expression patterns of non-viral transfection with GFP in the organ of Corti in vitro and in vivo. Gene therapy of the inner ear with non-viral vectors].

PubMed

Praetorius, M; Pfannenstiel, S; Klingmann, C; Baumann, I; Plinkert, P K; Staecker, H

2008-05-01

Diseases of the inner ear such as presbycusis, tinnitus, sudden hearing loss, and vertigo affect many patients, but so far there are no specific therapy options. Gene therapy might become a potential modality of treatment. Viral vectors are standard in animal models to date. Future considerations, however, call for a further evaluation of non-viral transfection methods. The non-viral transfection agents Metafectene, Superfect, Effectene, and Mirus TransIT were incubated with a plasmid coding for GFP. In vivo, the plasmid-agent mix was injected via the membrane of the round window, and 48 h later the inner ear was perfused, harvested, decalcified, and histologically evaluated for GFP expression. Cationic lipids (Metafectene) and dendrimers (Superfect) were able to transfect cells in the area of the organ of Corti and lead to GFP expression. The polyamine (Mirus TransIT) did show expression of GFP in the area of Rosenthal's canal and in the area of the inner hair cell. The combination of a non-liposomal lipid with a DNA condensing component (Effectene) did not show transfection of the organ of Corti. In the area of the spiral ganglia cells, GFP expression was seen with all the transfection agents. Non-viral transfection agents are able to introduce a reporter gene in cells of the inner ear in vitro and in vivo. There are, however, differences in the efficiency of the transfection. They might be an alternative in gene therapy of the inner ear. Further investigations to elucidate their potential are needed.

Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

PubMed

Magro, Massimiliano; Martinello, Tiziana; Bonaiuto, Emanuela; Gomiero, Chiara; Baratella, Davide; Zoppellaro, Giorgio; Cozza, Giorgio; Patruno, Marco; Zboril, Radek; Vianello, Fabio

2017-11-01

Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection. Copyright © 2017 Elsevier B.V. All rights reserved.

Attempts on producing lymphoid cell line from Penaeus monodon by induction with SV40-T and 12S EIA oncogenes.

PubMed

Puthumana, Jayesh; Prabhakaran, Priyaja; Philip, Rosamma; Singh, I S Bright

2015-12-01

In an attempt of in vitro transformation, transfection mediated expression of Simian virus-40 (T) antigen (SV40-T) and transduction mediated expression of Adenovirus type 12 early region 1A (12S E1A) oncogene were performed in Penaeus monodon lymphoid cells. pSV3-neo vector encoding SV40-T oncogene and a recombinant baculovirus BacP2-12S E1A-GFP encoding 12S E1A oncogene under the control of hybrid promoters were used. Electroporation and lipofection mediated transformation of SV40-T in lymphoid cells confirmed the transgene expression by phenotypic variation and the expression of GFP in co-transfection experiment. The cells transfected by lipofection (≥ 5%) survived for 14 days with lower toxicity (30%), whilst on electroporation, most of the cells succumbed to death (60%) and survived cells lived up to 7 days. Transduction efficiency in primary lymphoid cells was more than 80% within 14 days of post-transduction, however, an incubation period of 7 days post-transduction was observed without detectable expression of 12S E1A. High level of oncogenic 12S E1A expression were observed after 14 day post-transduction and the proliferating cells survived for more than 90 days with GFP expression, however, without in vitro transformation and immortalization. The study put forth the requirement of transduction mediated 'specific' oncogene expression along with telomerase activation and epigenetic induction for the immortalization and establishment of shrimp cell line. Copyright © 2015. Published by Elsevier Ltd.

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

Characterization of MUDENG, a novel anti-apoptotic protein

PubMed Central

Choi, J-H; Lim, J-B; Wickramanayake, D D; Wagley, Y; Kim, J; Lee, H-C; Seo, H G; Kim, T-H; Oh, J-W

2016-01-01

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death signaling. Astrocytes, the major glial cell type in the central nervous system, are a source of brain tumors. In this study, we examined MuD expression and function in human astroglioma cells. Stimulation of U251-MG cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a 40% decrease in cell viability and a 33% decrease in MuD protein levels, although not in MuD mRNA levels. To study the functional relevance of MuD expression, stable transfectants expressing high levels of MuD were generated. Stimulation of these transfectants with TRAIL resulted in enhanced cell survival (77% for stable and 46% for control transfectants). Depletion of MuD led to a marked reduction upon TRAIL stimulation in cell viability (22% in MuD-depleted cells and 54% in control cells). In addition, we observed that MuD depletion increased the susceptibility of the cells to TRAIL by enhancing the cleavage of caspase-3/-9 and BH3-interacting domain death agonist (Bid). A unique 25-kDa fragment of B-cell lymphoma 2 (Bcl-2) lacking BH4 was observed 60–180 min post TRAIL treatment in MuD-depleted cells, suggesting that Bcl-2 is converted from its anti-apoptotic form to the truncated pro-apoptotic form. Importantly, the TRAIL-mediated decrease in cell viability in MuD-depleted cells was abrogated upon Bid depletion, indicating that the role of MuD in apoptotic signaling takes place at the Bid and Bcl-2 junction. MuD localizes predominantly in the endoplasmic reticulum and partly in the mitochondria and its amounts are reduced 6 h post TRAIL stimulation, presumably via caspase-3-mediated MuD cleavage. Collectively, these results suggest that MuD, a novel signaling protein, not only possesses an anti-apoptotic function but may also constitute an important target for the design of ideal candidates for combinatorial treatment strategies for glioma cells. PMID:27136675

[Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma].

PubMed

Zhang, Ling; He, Chanting; Bi, Yanghui; Liu, Feng; Cui, Heyang; Wang, Juan; Song, Bin; Shi, Ruyi; Yang, Bin; Wang, Fang; Jia, Zhiwu; Zhao, Zhenxiang; Liu, Jing

2015-04-01

To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma. Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene

PubMed Central

Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Chun, Changho; Kwon, Young Jik; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

2014-01-01

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments. PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect. PMID:24610460

Magnetofection™ of NMDA Receptor Subunits GluN1 and GluN2A Expression Vectors in Non-Neuronal Host Cells.

PubMed

Bruneau, Nadine; Szepetowski, Pierre

2017-01-01

The functional study of reconstituted NMDA receptors (NMDARs) in host cells requires that the corresponding vectors for the expression of the NMDAR subunits are co-transfected with high efficiency. Magnetofection™ is a technology used to deliver nucleic acids to cells. It is driven and site-specifically guided by the attractive forces of magnetic fields acting on magnetic nanoparticles that are associated with nucleic acid vectors. In magnetofection™, cationic lipids form self-assembled complexes with the nucleic acid vectors of interest. Those complexes are then associated with magnetic nanoparticles that are concentrated at the surface of cultured cells by applying a permanent magnetic field. Magnetofection™ is a simple method to transfect cultured cells with high transfection rates. Satisfactory expression levels are obtained with very low amounts of nucleic acid vector. Moreover, incubation time with host cells is less than 1 h, as compared with the several hours needed with standard transfection assays.

Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

NASA Astrophysics Data System (ADS)

Kamau Chapman, Sarah W.; Hassa, Paul O.; Koch-Schneidemann, Sabine; von Rechenberg, Brigitte; Hofmann-Amtenbrink, Margarethe; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hottiger, Michael O.

Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

TRPV2 channel negatively controls glioma cell proliferation and resistance to Fas-induced apoptosis in ERK-dependent manner.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Amantini, Consuelo; Farfariello, Valerio; Ricci-Vitiani, Lucia; Caprodossi, Sara; Arcella, Antonella; Santoni, Matteo; Giangaspero, Felice; De Maria, Ruggero; Santoni, Giorgio

2010-05-01

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 2 (TRPV2) in human glioma cells. By Real-Time-PCR and western blot analysis, we found that TRPV2 messenger RNA (mRNA) and protein were expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased (n = 49 cases), and in U87MG cells and in MZC, FCL and FSL primary glioma cells. To investigate the function of TRPV2 in glioma, small RNA interfering was used to silence TRPV2 expression in U87MG cells. As evaluated by RT-Profiler PCR array, siTRPV2-U87MG transfected cells displayed a marked downregulation of Fas and procaspase-8 mRNA expression, associated with upregulation of cyclin E1, cyclin-dependent kinase 2, E2F1 transcriptor factor 1, V-raf-1 murine leukemia viral oncogene homolog 1 and Bcl-2-associated X protein (Bcl-X(L)) mRNA expression. TRPV2 silencing increased U87MG cell proliferation as shown by the increased percentage of cells incorporating 5-bromo-2-deoxyuridine expressing beta(III)-tubulin and rescued glioma cells to Fas-induced apoptosis. These events were dependent on extracellular signal-regulated kinase (ERK) activation: indeed inhibition of ERK activation in siTRPV2-U87MG transfected cells by treatment with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, reduced Bcl-X(L) protein levels, promoted Fas expression, and restored Akt/protein kinase B pathway activation leading to reduced U87MG cell survival and proliferation, and increased sensitivity to Fas-induced apoptosis. In addition, transfection of TRPV2 in MZC glioma cells, by inducing Fas overexpression, resulted in a reduced viability and an increased spontaneous and Fas-induced apoptosis. Overall, our findings indicate that TRPV2 negatively controls glioma cell survival and proliferation, as well as resistance to Fas-induced apoptotic cell death in an ERK-dependent manner.

Transforming growth factor beta1 gene variation Leu10Pro affects secretion and function in hepatic cells.

PubMed

Gu, Xing; Ji, Xin; Shi, Le-Hua; Yi, Chang-Hong; Zhao, Yun-Peng; Wang, Ai-Hua; Lu, Lun-Gen; Yu, Wen-Bo; Gao, Chun-Fang

2012-11-01

Our previous work revealed transforming growth factor beta1 (TGFβ1) gene polymorphisms are associated with susceptibility to hepatocellular carcinoma and liver cirrhosis. However, no further study of functional substitution in hepatic cells has yet been reported. This study was designed to uncover the functional mechanisms of TGFβ1 gene polymorphisms in the pathogenesis of liver diseases. Two recombinant TGFβ1 expression plasmids containing TGFβ1 codon 10 Leu/Pro variation were constructed with CMV promoter and transfected into human hepatoma cell lines (HepG2 and SMMU 7721), hepatic stellate cells (LX-2), and immortalized hepatocytes (L02). The secretion capacities of TGFβ1 protein in the transfected cells were determined by ELISA. Apoptosis, proliferative activity, and expression of CD 105, CD83, and CD80 were also measured by use of flow cytometry. The ELISA results showed that cells transfected with CMV-Pro10 were more capable of TGFβ1 secretion than those transfected with CMV-Leu10. Functionally, CMV-Pro10 was more apoptosis-protective and induced more proliferation than CMV-Leu10 in transfected hepatic cells. Pro10 up-regulated expression of CD105 and down-regulated expression of CD83. TGFβ1 gene Leu10Pro variation in signal peptide has significant effects on TGFβ1 secretion and functions in hepatic cells.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed

Blochlinger, K; Diggelmann, H

1984-12-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed Central

Blochlinger, K; Diggelmann, H

1984-01-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells. Images PMID:6098829

Digital optical imaging of green fluorescent proteins for tracking vascular gene expression: feasibility study in rabbit and human cell models.

PubMed

Yang, X; Liu, H; Li, D; Zhou, X; Jung, W C; Deans, A E; Cui, Y; Cheng, L

2001-04-01

To investigate the feasibility of using a sensitive digital optical imaging technique to detect green fluorescent protein (GFP) expressed in rabbit vasculature and human arterial smooth muscle cells. A GFP plasmid was transfected into human arterial smooth muscle cells to obtain a GFP-smooth muscle cell solution. This solution was imaged in cell phantoms by using a prototype digital optical imaging system. For in vivo validation, a GFP-lentivirus vector was transfected during surgery into the carotid arteries of two rabbits, and GFP-targeted vessels were harvested for digital optical imaging ex vivo. Optical imaging of cell phantoms resulted in a spatial resolution of 25 microm/pixel. Fluorescent signals were detected as diffusely distributed bright spots. At ex vivo optical imaging of arterial tissues, the average fluorescent signal was significantly higher (P <.05) in GFP-targeted tissues (mean +/- SD, 9,357.3 absolute units of density +/- 1,001.3) than in control tissues (5,633.7 absolute units of density +/- 985.2). Both fluorescence microscopic and immunohistochemical findings confirmed these differences between GFP-targeted and control vessels. The digital optical imaging system was sensitive to GFPs and may potentially provide an in vivo imaging tool to monitor and track vascular gene transfer and expression in experimental investigations.

Recent advances in magnetofection and its potential to deliver siRNAs in vitro.

PubMed

Mykhaylyk, Olga; Zelphati, Olivier; Hammerschmid, Edelburga; Anton, Martina; Rosenecker, Joseph; Plank, Christian

2009-01-01

This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for magnetofection and the association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by magnetofection in every cell type.

Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous beta-TCP ceramic scaffolds.

PubMed

Guo, Xiaodong; Zheng, Qixin; Kulbatski, Iris; Yuan, Quan; Yang, Shuhua; Shao, Zengwu; Wang, Hong; Xiao, Baojun; Pan, Zhengqi; Tang, Shuo

2006-09-01

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous beta tricalcium phosphate (beta-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

Role of cholesterol on the transfection barriers of cationic lipid/DNA complexes

NASA Astrophysics Data System (ADS)

Pozzi, Daniela; Cardarelli, Francesco; Salomone, Fabrizio; Marchini, Cristina; Amenitsch, Heinz; Barbera, Giorgia La; Caracciolo, Giulio

2014-08-01

Most lipid formulations need cholesterol for efficient transfection, but the precise motivation remains unclear. Here, we have investigated the effect of cholesterol on the transfection efficiency (TE) of cationic liposomes made of 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphocholine in Chinese hamster ovary cells. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by TE, synchrotron small angle X-ray scattering, and laser scanning confocal microscopy experiments. We prove that cholesterol-containing lipoplexes enter the cells using different endocytosis pathways. Formulations with high cholesterol content efficiently escape from endosomes and exhibit a lamellar-nonlamellar phase transition in mixture with biomembrane mimicking lipid formulations. This might explain both the DNA release ability and the high transfection efficiency. These studies highlight the enrichment in cholesterol as a decisive factor for transfection and will contribute to the rational design of lipid nanocarriers with superior TE.

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

2015-08-01

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

Solid lipid nanoparticles release DNA upon endosomal acidification in human embryonic kidney cells.

PubMed

Radaic, A; de Jesus, M B

2018-08-03

Nanotechnology can produce materials with unique features compared to their bulk counterparts, which can be useful for medical applications (i.e. nanomedicine). Among the therapeutic agents used in nanomedicine, small molecules or biomacromolecules, such as proteins or genetic materials, can be designed for disease diagnostics and treatment. To transport these biomacromolecules to the target cells, nanomedicine requires nanocarriers. Solid lipid nanoparticles (SLNs) are among the promising nanocarriers available, because they can be made from biocompatible materials and present high stability (over one year). In addition, upon the binding genetic material, SLNs form SLNplexes. However, little is yet known about how cells process these SLNplexes-in particular, how internalization and endosome acidification affects the transfection mediated by SLNplexes. Therefore, we aim to investigate how these processes affect SLNplex transfection in HEK293T cells. We find that the SLNplex is mainly internalized by clathrin-mediated endocytosis, which is a fast and reliable pathway to transfection, leading to approximately 60% transfection efficiency. Interestingly, upon acidification (below pH 5.0), the SLN seems to release its DNA content, which can be an essential step for SLNplex transfection. The underlying mechanisms described in this work may help improve SLNplex formulations and transfection efficiency. Moreover, these advances can improve the field of nanomedical research and bring new ways to cure diseases.

Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

NASA Astrophysics Data System (ADS)

Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

2014-03-01

Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

DEVELOPMENT OF AN ENVIRONMENTAL ESTROGEN SCREEN USING TRANSIENTLY TRANSFECTED RAINBOW TROUT CELL LINES

EPA Science Inventory

Rainbow troutp hepatoma (RTH-149) and gonad cells (RTG-2) were used to develop a screening protocol for estrogen disrupting chemicals. Transfection of an estrogen-responsive luciferase reporter plasmid into...

RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration.

PubMed

Wu, Dongni; Zhang, Yongnu; Xu, Xiaoting; Guo, Ting; Xie, Deming; Zhu, Rong; Chen, Shengfeng; Ramakrishna, Seeram; He, Liumin

2018-05-01

In this study, we prepared a multifunctional gene delivery nanovector containing a chitosan (CS) backbone and polyethylenimine (PEI) arms with arginine-glycine-aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). Branched PEI, with a molecular weight of 2000 Da, was used to achieve a balance between biocompatibility and transfection efficiency, whereas RGD/TAT peptides were conjugated for enhanced targeting ability and cellular uptake. Synthesis of the copolymers was confirmed by characterizing the chemical structure with 1 H nuclear magnetic resonance and Fourier Transform Infrared Spectroscopy (FTIR). The nanovector was biocompatible with cells and showed excellent capability for DNA condensation; the resulting complexes with DNA were well-formed, and possessed small particle size and reasonable positive charge. Higher gene transfection efficiency, compared to that achieved with PEI (25 kDa), was confirmed in tumor (HeLa cells) and normal cells (293T and NIH 3T3 cells). More importantly, the cells transfected with the chitosan-graft-PEI-PEG/pCMV-EGFP-Ntf3 complex produced sustained neurotrophin-3 with a linear increase in cumulative concentration, which induced neuronal differentiation of neural stem cell and promoted neurite outgrowth. These findings suggested that our multifunctional copolymers might be ideal nanovectors for engineering cells via gene transfection, and could potentially be applied in tumor therapy and regenerative medicine. We successfully prepared a multifunctional gene delivery nanovector containing branched PEI with a molecular weight of 2000 Da to balance between biocompatibility and transfection efficiency, and RGD/TAT peptides for enhanced targeting ability and cellular uptake. The well-formed CPPP/DNA complexes of small particle size and reasonable positive charges potentially enhanced gene transfection in both tumor and normal cells. More importantly, the CPPP/pCMV-EGFP-Ntf3 complex-transfected 293T cells could produce sustained NT-3 with a constant ratio, which induced neuron differentiation of NSC and promoted neurite outgrowth. Therefore, our study provided an effective strategy for producing neurotrophins by engineering cells with gene delivery, which deserved wide investigation and potential application in regenerative medicine. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Inhibition of apoptosis by p26: implications for small heat shock protein function during Artemia development.

PubMed

Villeneuve, Tania S; Ma, Xiaocui; Sun, Yu; Oulton, Mindy M; Oliver, Ann E; MacRae, Thomas H

2006-01-01

p26, an abundantly expressed small heat shock protein, is thought to establish stress resistance in oviparously developing embryos of the crustacean Artemia franciscana by preventing irreversible protein denaturation, but it might also promote survival by inhibiting apoptosis. To test this possibility, stably transfected mammalian cells producing p26 were generated and their ability to resist apoptosis induction determined. Examination of immunofluorescently stained transfected 293H cells by confocal microscopy demonstrated p26 is diffusely distributed in the cytoplasm with a minor amount of the protein in nuclei. As shown by immunoprobing of Western blots, p26 constituted approximately 0.6% of soluble cell protein. p26 localization and quantity were unchanged during prolonged culture, and the protein had no apparent ill effects on transfected cells. Molecular sieve chromatography in Sepharose 6B revealed p26 oligomers of about 20 monomers, with a second fraction occurring as larger aggregates. A similar pattern was observed in sucrose gradients, but overall oligomer size was smaller. Mammalian cells containing p26 were more thermotolerant than cells transfected with the expression vector only, and as measured by annexin V labeling, Hoescht 33342 nuclear staining and procaspase-3 activation, transfected cells effectively resisted apoptosis induction by heat and staurosporine. The ability to confer thermotolerance and limit heat-induced apoptosis is important because Artemia embryos are frequently exposed to high temperature in their natural habitat. p26 also blocked apoptosis in transfected cells during drying and rehydration, findings with direct relevance to Artemia life history characteristics because desiccation terminates cyst diapause. Thus, in addition to functioning as a molecular chaperone, p26 inhibits apoptosis, an activity shared by other small heat shock proteins and with the potential to play an important role during Artemia embryo development.

Clofibric acid induces hepatic CYP 2B1/2 via constitutive androstane receptor not via peroxisome proliferator activated receptor alpha in rat.

PubMed

Ibrahim, Zein Shaban; Ahmed, Mohamed Mohamed; El-Shazly, Samir Ahmed; Ishizuka, Mayumi; Fujita, Shoichi

2014-01-01

Peroxisome proliferator activated receptor α (PPARα) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPARα were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPARα transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPARα.

Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells.

PubMed

Hlawaty, Hanna; San Juan, Aurélie; Jacob, Marie-Paule; Vranckx, Roger; Letourneur, Didier; Feldman, Laurent J

2007-12-01

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in approximately 80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 +/- 19%, 43 +/- 14%, and 36 +/- 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs (P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries (P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.

β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

PubMed Central

Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P.; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-01-01

Hypoxia-inducible factor-1α (HIF-1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF-1α remain unclear. β-catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF-1α and β-catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4-2B, were grouped as follows: Negative control (no treatment), HIF-1α overexpression group (transfected with HIF-1α overexpression plasmid) and β-catenin silenced group (transfected with HIF-1α plasmids and β-catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4-2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4-2B cells, transfection with HIF-1α overexpression plasmid led to an enhanced β-catenin nuclear translocation, while β-catenin silencing inhibited β-catenin nuclear translocation. The enhanced β-catenin nuclear translocation induced by HIF-1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non-homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF-1α overexpression enhanced β-catenin nuclear translocation, which led to the activation of the β-catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF-1α overexpression promotes the radioresistance of PCa cells. PMID:29658569

Up-regulation of VEZT by small activating RNA inhibits the proliferation, invasion and migration of gastric cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Detian; Shang, Liang; Peng, Lipan

Objective: To identify an effective saRNA sequence that can specifically up-regulate VEZT expression and to determine the influence of saRNA had on gastric cancer cell growth, proliferation, invasion and migration. Methods: Three various saRNAs, that target the VEZT gene promoter at different locations relative to the transcription start site were synthesized. A dsControl saRNA was synthesized as a negative control, and a specific shRNA was synthesized to knockdown VEZT and eliminate any off-target effects of the saRNA. Both SGC-7901 and M-28 cells were either transfected with the different saRNAs, or treated with Lipofectamine2000 alone. To determine the most effective saRNA, real-timemore » PCR and Western blot were used to determine the VEZT mRNA and protein content, respectively, of each treatment group. After selection, both cell lines were treated with the chosen saRNA, dsControl or Lipofectamine2000. The saRNA treated cells were divided into two groups: the first group was used immediately in the experiments, and the second group was transfected with shRNA by using RNAi-Mate. The proliferation of cells transfected with saRNA, or saRNA and shRNA, as well as the other control cells, was detected by CCK-8. The invasive and migratory abilities were determined using the transwell chamber assay. Results: We identified the most effective saRNA via real-time PCR and Western blot. The selected saRNA inhibited the growth, invasion and migration of GC cells by specially reactivating VEZT. The real-time PCR and Western blot results showed that treatment with saRNA caused a significant up-regulation of VEZT, and an obvious decrease in the proliferative, invasive and migratory abilities; compared with the control groups (P < 0.01); furthermore, there were no significant differences among the control groups (P > 0.05). This phenomenon provides a theoretical basis for saRNA design and gene therapy for gastric cancer. - Highlights: • saRNA is a brand-new type of small non-coding RNA. It provides a brand-new therapeutic tool for gastric cancer. • In this manuscript, we designed the new saRNA that was effective for VEZT for the first time. • VEZT is a new TSG, and the reactivation through saRNA is never reported before. • It's the first time to report the RNAa in gastric cancer.« less

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles.

PubMed

Lee, Jyun-Lin; Lo, Chia-Wen; Ka, Shuk-Man; Chen, Ann; Chen, Wen-Shiang

2012-05-30

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

PubMed

Narayanan, Sharmila; Sanpui, Pallab; Sahoo, Lingaraj; Ghosh, Siddhartha Sankar

2016-10-01

In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Heterologous Expression of Gene of Interest Using the Marine Protozoan Perkinsus marinus

NASA Astrophysics Data System (ADS)

Cold, E. R.

2016-02-01

Perkinsus marinus is a marine protozoan parasite that causes "Dermo" disease in eastern oysters (Crassostrea virginica). P. marinus is closely related to Plasmodium falciparum which causes malaria. A recent study has showed that P. marinus causes no pathology damage but an immune response in humanized mouse, providing the bases for a genetically modified P. marinus expressing Plasmodium genes to be used as a vaccination delivery system for malaria and other pathogenic diseases. A modified plasmid vector (pMOE-GFP) based on highly expressed gene tagged with green fluorescence protein and targeted to P. marinus cell wall was used to clone MSP8 and HAP2. MSP8 encodes for merozoite surface in P. falciparum and HAP2 is essential for fusion of male and female gametes; genetic disruption of the HAP2 locus revealed that parasite fertilization is prevented. Using electroporation, MSP8 and HAP2 plasmid were introduced into the P. marinus trophozoites. As controls pMOE-GFP was transfected into P. mediterraneus, P. atlanticus and P. chesapeaki. Transfection conditions included 5x107 Perkinsus trophozoites and 10 µg of plasmid using Nucleofector® technology (D-023 program). The cells were recovered in 3 mL of Perkinsus culture media and transfected trophozoites were examined for green fluorescence. To facilitate subcloning of cells expressing GFP, we optimized a DME: HAM's F12 -5% FBS -containing agar solid medium for plating Perkinsus. Examination of all transfected cells indicates expression of both MSP8 and HAP2. This is the first time that genes of a protozoan parasite have been expressed in a marine protozoan. It was also concluded that P. mediterraneus, P. atlanticus and P. chesapeaki were stable mutation and can be isolated for further research.

MET signalling in primary colon epithelial cells leads to increased transformation irrespective of aberrant Wnt signalling

PubMed Central

Boon, E M J; Kovarikova, M; Derksen, P W B; van der Neut, R

2005-01-01

It has been shown that in hereditary and most sporadic colon tumours, components of the Wnt pathway are mutated. The Wnt target MET has been implicated in the development of colon cancer. Here, we show that overexpression of wild-type or a constitutively activated form of MET in colon epithelial cells leads to increased transformation irrespective of Wnt signalling. Fetal human colon epithelial cells without aberrant Wnt signalling were transfected with wild-type or mutated MET constructs. Expression of these constructs leads to increased phosphorylation of MET and its downstream targets PKB and MAPK. Upon stimulation with HGF, the expression of E-cadherin is downregulated in wild-type MET-transfected cells, whereas cells expressing mutated MET show low E-cadherin levels independent of stimulation with ligand. This implies a higher migratory propensity of these cells. Furthermore, fetal human colon epithelial cells expressing the mutated form of MET have colony-forming capacity in soft agar, while cells expressing wild-type MET show an intermediate phenotype. Subcutaneous injection of mutated MET-transfected cells in nude mice leads to the formation of tumours within 12 days in all mice injected. At this time point, mock-transfected cells do not form tumours, while wild-type MET-transfected cells form subcutaneous tumours in one out of five mice. We thus show that MET signalling can lead to increased transformation of colon epithelial cells independent of Wnt signalling and in this way could play an essential role in the onset and progression of colorectal cancer. PMID:15785735

Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

PubMed Central

Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

2011-01-01

Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

PubMed Central

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2 × 108 pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application. PMID:17225867

Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

PubMed

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application.

A Direct Synergistic Effect of Immunotherapy and Chemotherapy as a New Paradigm in Treatment of Breast Cancer

DTIC Science & Technology

2010-04-01

mouse macrophage nucleofector kit (Program-Y-01) was used. For EL4 cells mouse cell transfection kit (Program-C-09) was used. As controls...direct synergy between immunotherapy and chemotherapy in vitro. We found that pre-treatment of tumor target cells with doxorubicin or paclitaxel...significantly increased cytotoxic effect of T-lymphocytes. Importantly, that effect was antigen-specific, since it was observed only in tumor cells loaded

Size, charge and concentration dependent uptake of iron oxide particles by non-phagocytic cells.

PubMed

Thorek, Daniel L J; Tsourkas, Andrew

2008-09-01

A promising new direction for contrast-enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of superparamagnetic iron oxide (SPIO)-labeled cells in vivo. Despite the large number of cell labeling studies that have been performed with SPIO particles of differing size and surface charge, it remains unclear which SPIO configuration provides optimal contrast in non-phagocytic cells. This is largely because contradictory findings have stemmed from the variability and imprecise control over surface charge, the general need and complexity of transfection and/or targeting agents, and the limited number of particle configurations examined in any given study. In the present study, we systematically evaluated the cellular uptake of SPIO in non-phagocytic T cells over a continuum of particle sizes ranging from 33nm to nearly 1.5microm, with precisely controlled surface properties, and without the need for transfection agents. SPIO labeling of T cells was analyzed by flow cytometry and contrast enhancement was determined by relaxometry. SPIO uptake was dose-dependent and exhibited sigmoidal charge dependence, which was shown to saturate at different levels of functionalization. Efficient labeling of cells was observed for particles up to 300nm, however, micron-sized particle uptake was limited. Our results show that an unconventional highly cationic particle configuration at 107nm maximized MR contrast of T cells, outperforming the widely utilized USPIO (<50nm).

Size, Charge and Concentration Dependent Uptake of Iron Oxide Particles by Non-Phagocytic Cells

PubMed Central

Thorek, Daniel L.J.; Tsourkas, Andrew

2008-01-01

A promising new direction for contrast-enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of superparamagnetic iron oxide (SPIO)-labeled cells in vivo. Despite the large number of cell labeling studies that have been performed with SPIO particles of differing size and surface charge, it remains unclear which SPIO configuration provides optimal contrast in non-phagocytic cells. This is largely because contradictory findings have stemmed from the variability and imprecise control over surface charge, the general need and complexity of transfection and/or targeting agents, and the limited number of particle configurations examined in any given study. In the present study, we systematically evaluated the cellular uptake of SPIO in non-phagocytic T cells over a continuum of particle sizes ranging from 33 nm to nearly 1.5 μm, with precisely controlled surface properties, and without the need for transfection agents. SPIO labeling of T cells was analyzed by flow cytometry and contrast enhancement was determined by relaxometry. SPIO uptake was dose dependent and exhibited sigmoidal charge dependence, which was shown to saturate at different levels of functionalization. Efficient labeling of cells was observed for particles up to 300nm, however micron-sized particle uptake was limited. Our results show that an unconventional highly cationic particle configuration at 107 nm maximized MR contrast of T cells, outperforming the widely utilized USPIO (<50 nm). PMID:18533252

Targeted nanobubbles in low-frequency ultrasound-mediated gene transfection and growth inhibition of hepatocellular carcinoma cells.

PubMed

Wu, Bolin; Qiao, Qiang; Han, Xue; Jing, Hui; Zhang, Hao; Liang, Hongjian; Cheng, Wen

2016-09-01

The use of SonoVue combined with ultrasound exposure increases the transfection efficiency of short interfering RNA (siRNA). The objective of this study was to prepare targeted nanobubbles (TNB) conjugated with NET-1 siRNA and an antibody GPC3 to direct nanobubbles to hepatocellular carcinoma cells. SMMC-7721 human hepatocellular carcinoma cells were treated with six different groups. The transfection efficiency and cellular apoptosis were measured by flow cytometry. The protein and messenger RNA (mRNA) expression were measured by Western blot and quantitative real-time PCR, respectively. The migration and invasion potential of the cells were determined by Transwell analysis. The results show that US-guided siRNA-TNB transfection effectively enhanced gene silencing. In summary, siRNA-TNB may be an effective delivery vector to mediate highly effective RNA interference in tumor treatment.

The Role of Calgranulin Overexpression in Breast Cancer Progression

DTIC Science & Technology

2005-09-01

transfected cells. As shown in Figure 4, exposure of MCF-7 cells to 25ng/ml OSM for 24 hours caused a very significant increase in Cal A levels . Interestingly...Cal A expression was not observed in the parental or vector alone cells, but the transfected cells showed an elevation in Cal A levels . The

Overexpression of c-Met in bone marrow mesenchymal stem cells improves their effectiveness in homing and repair of acute liver failure.

PubMed

Wang, Kun; Li, Yuwen; Zhu, Tiantian; Zhang, Yongting; Li, Wenting; Lin, Wenyu; Li, Jun; Zhu, Chuanlong

2017-07-05

Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has emerged as a novel therapy for acute liver failure (ALF). However, the homing efficiency of BMSCs to the injured liver sites appears to be poor. In this study, we aimed to determine if overexpression of c-Met in BMSCs could promote the homing ability of BMSCs to rat livers affected by ALF. Overexpression of c-Met in BMSCs (c-Met-BMSCs) was attained by transfection of naive BMSCs with the lenti-c-Met-GFP. The impact of transplanted c-Met-BMSCs on both homing and repair of ALF was evaluated and compared with lenti-GFP empty vector transfected BMSCs (control BMSCs). After cells were transfected with the lenti-c-Met-GFP vector, the BMSCs displayed very high expression of c-Met protein as demonstrated by Western blot. In addition, in vitro transwell migration assays showed that the migration ability of c-Met-BMSCs was significantly increased in comparison with that of control BMSCs (P < 0.05), and was dependent on hepatocyte growth factor (HGF). Furthermore, rats with ALF that received transplanted c-Met-BMSCs showed significantly improved homing ability to the injured liver; this was accompanied by elevated survival rates and liver function in the ALF rats. Parallel pathological examination further confirmed that transplantation of c-Met-BMSCs ameliorated liver injury with reduced hepatic activity index (HAI) scores, and that the effects of c-Met-BMSCs were more profound than those of control BMSCs. Overexpression of c-Met promotes the homing of BMSCs to injured hepatic sites in a rat model of ALF, thereby improving the efficacy of BMSC therapy for ALF repair.

Downregulation of microRNA‑574 in cancer stem cells causes recurrence of prostate cancer via targeting REL.

PubMed

Lai, Xiaodong; Guo, Yanchun; Guo, Zhitao; Liu, Ruibao; Wang, Xunguo; Wang, Fang

2016-12-01

miR‑574‑5p has been reported involved in the pathogenesis of numerous human malignancies such as colorectal and lung cancer. In this study, we aimed to explore the roles of REL and miR‑574 in the recurrence of prostate cancer (PCa) and to identify the underlying molecular mechanisms. Our literature search found that miR‑574 is regulated in cancer stem cells (CSCs), and next we used the microRNA (miRNA) database (www.mirdb.org) to find REL as a target of miR‑574. Luciferase assay was performed to verify the miRNA/target relationship. Oligo-transfection, real‑time PCR and western blot analysis were used to support the conclusions. We validated REL to be the direct gene via luciferase reporter assay system, and real‑time PCR and western blot analysis were also conducted to study the mRNA and protein expression level of REL between different groups (recurrence and non‑recurrence) or cells treated with scramble control, miR‑574 mimics, REL siRNA and miR‑574 inhibitors, indicating the negative regulatory relationship between miR‑574 and REL. We also investigated the relative viability of prostate CSCs when transfected with scramble control, miR‑574 mimics, REL siRNA and miR‑574 inhibitors to validate miR‑574 to be positively interfering with the viability of prostate CSCs. We then investigated the relative apoptosis of prostate CSCs when transfected with scramble control, miR‑574 mimics, REL siRNA and miR‑574 inhibitors. The results showed miR‑574 inhibited apoptosis. In conclusion, miR‑574 might be a novel prognostic and therapeutic target in the management of PCa recurrence.

Familial cancer associated with a polymorphism in ARLTS1.

PubMed

Calin, George Adrian; Trapasso, Francesco; Shimizu, Masayoshi; Dumitru, Calin Dan; Yendamuri, Sai; Godwin, Andrew K; Ferracin, Manuela; Bernardi, Guido; Chatterjee, Devjani; Baldassarre, Gustavo; Rattan, Shashi; Alder, Hansjuerg; Mabuchi, Hideaki; Shiraishi, Takeshi; Hansen, Lise Lotte; Overgaard, Jens; Herlea, Vlad; Mauro, Francesca Romana; Dighiero, Guillaume; Movsas, Benjamin; Rassenti, Laura; Kipps, Thomas; Baffa, Raffaele; Fusco, Alfredo; Mori, Masaki; Russo, Giandomenico; Liu, Chang-Gong; Neuberg, Donna; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M

2005-04-21

The finding of hemizygous or homozygous deletions at band 14 on chromosome 13 in a variety of neoplasms suggests the presence of a tumor-suppressor locus telomeric to the RB1 gene. We studied samples from 216 patients with various types of sporadic tumors or idiopathic pancytopenia, peripheral-blood samples from 109 patients with familial cancer or multiple cancers, and control blood samples from 475 healthy people or patients with diseases other than cancer. We performed functional studies of cell lines lacking ARLTS1 expression with the use of both the full-length ARLTS1 gene and a truncated variant. We found a gene at 13q14, ARLTS1, a member of the ADP-ribosylation factor family, with properties of a tumor-suppressor gene. We analyzed 800 DNA samples from tumors and blood cells from patients with sporadic or familial cancer and controls and found that the frequency of a nonsense polymorphism, G446A (Trp149Stop), was similar in controls and patients with sporadic tumors but was significantly more common among patients with familial cancer than among those in the other two groups (P=0.02; odds ratio, 5.7; 95 percent confidence interval, 1.3 to 24.8). ARLTS1 was down-regulated by promoter methylation in 25 percent of the primary tumors we analyzed. Transfection of wild-type ARLTS1 into A549 lung-cancer cells suppressed tumor formation in immunodeficient mice and induced apoptosis, whereas transfection of truncated ARLTS1 had a limited effect on apoptosis and tumor suppression. Microarray analysis revealed that the wild-type and Trp149Stop-transfected clones had different expression profiles. A genetic variant of ARLTS1 predisposes patients to familial cancer. Copyright 2005 Massachusetts Medical Society.

Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice.

PubMed

Suzuki, Jun-ichi; Ogawa, Masahito; Takayama, Kiyoshi; Taniyama, Yoshiaki; Morishita, Ryuichi; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

2010-03-02

The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases. Copyright 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Large-Scale Transient Transfection of Chinese Hamster Ovary Cells in Suspension.

PubMed

Rajendra, Yashas; Balasubramanian, Sowmya; Hacker, David L

2017-01-01

We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 10 6 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

Selective Gene Transfection of Individual Cells In Vitro with Plasmonic Nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina; Samaniego, Adam P.; Wen, Jianguo; Metelitsa, Leonid; Chang, Chung-Che; Lapotko, Dmitri

2011-01-01

Gene delivery and transfection of eukaryotic cells is widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. PMID:21315120

Selective gene transfection of individual cells in vitro with plasmonic nanobubbles.

PubMed

Lukianova-Hleb, Ekaterina Y; Samaniego, Adam P; Wen, Jianguo; Metelitsa, Leonid S; Chang, Chung-Che; Lapotko, Dmitri O

2011-06-10

Gene delivery and transfection of eukaryotic cells are widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+ CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of breast cancer metastasis by co-transfection of miR-31/193b-mimics

PubMed Central

Hashemi, Zahra Sadat; Moghadam, Mehdi Forouzandeh; Farokhimanesh, Samila; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2018-01-01

Objective(s): Various studies have been conducted to reduce the metastatic behavior of cancerous cells. In this regard, ectopic expression of anti-metastatic microRNAs by miR-mimic and miR-restoration-based therapies could bring new insights to the field. In the present study, the consequences of co-transfecting breast cancer cell lines with miR-193b and miR-31 were investigated via invasion and migration assays. Materials and Methods: Double stranded oligonucleotide of mature miR-193b-3p and miR-31-5p were cloned into pcDNA 6.2gw/EmGFP plasmid. The resulting plasmids were used for transfection. Real time-PCR was performed to assess the expression of miR-193b and miR-31 as well as Ras homolog gene family member A (RhoA) and urokinase-type plasminogen activator (uPA) as miR targets. Scratch, Transwell migration and Matrigel invasion assays were carried out to assess the extent of migration and invasion of cell lines. Results: The most significant increase in expression of miRs belonged to the single transfection of mimic-miRs in MDA-MB231. Although the co-transfection was not as successful as single transfection in miR expression, it was significantly more effective in inhibition of the cells invasive potential. Conclusion: Although the miR-restoration therapy based on co-transfection of two miRs could be less effective in expression of each miRNA, the resulting decrease in metastatic behavior of the cells is more significant due to collective effect of co-transfection to decrease target gene expression. Our results revealed that employing this sort of combinatorial strategies could lead to more efficient reduction in metastatic behavior. It seems that using this strategy would bring about more successful therapeutic outcomes. PMID:29796229

Activation and Inhibition of The Wnt3A Signaling Pathway in Buffalo (Bubalus bubalis) Embryonic Stem Cells: Effects of WNT3A, Bio and Dkk1.

PubMed

Zandi, Mohammad; Shah, Syed Mohamad; Muzaffar, Musharifa; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Sham Manik, Radhey; Chauhan, Manmohan Singh

2015-01-01

This research studies the effects of activation and inhibition of Wnt3A signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells. To carry on this experimental study, the effects of activation and inhibition of Wnt3A signaling in buffalo ES cell-like cells were examined using Bio (0.5 mM) combined with WNT3A (200 ng/ml), as an activator, and Dickkopf-1 (Dkk1, 250 ng/ml), as an inhibitor, of the pathway. ES cells were cultured up to three weeks in ES cell medium without fibroblast growth factor-2 (FGF-2) and leukemia inhibitory factor (LIF), but in the presence of Bio, WNT3A, Bio+WNT3A and Dkk1. The effects of these supplements were measured on the mean area of ES cell colonies and on the expression levels of a number of important genes related to pluripotency (Oct4, Nanog, Sox2 and c-Myc) and the Wnt pathway (β-catenin). ES cell colonies cultured in ES cell medium that contained optimized quantities of LIF and FGF-2 were used as the control. Data were collected for week-1 and week-3 treated cultures. In addition, WNT3A-transfected ES cells were compared with the respective mock-transfected colonies, either alone or in combination with Dkk1 for expression of β-catenin and the pluripotency-related genes. Data were analyzed by ANOVA, and statistical significance was accepted at P<0.05. Among various examined concentrations of Bio (0.5-5 mM), the optimum effect was observed at the 0.5 mM dose as indicated by colony area and expressions of pluripotency-related genes at both weeks-1 and -3 culture periods. At this concentration,the expressions of Nanog, Oct3/4, Sox2, c-Myc and β-catenin genes were nonsignificantly higher compared to the controls. Expressions of these genes were highest in the Bio+WNT3A treated group, followed by the WNT3A and Bio-supplemented groups, and lowest in the Dkk1-treated group. The WNT-transfected colonies showed higher expressions compared to both mock and Dkk1-treated mock transfected colonies. WNT3A functions to maintain the pluripotency of ES cell-like cells both as an exogenous growth factor as well as an endogenously expressed gene. It complements the absence of FGF-2 and LIF, otherwise propounded essential for buffalo ES cell culture. WNT3A antagonizes the inhibitory effects of Dkk1 and acts in combination with its activator, Bio, to activate the Wnt signaling pathway.

Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

PubMed Central

Reeves, R; Gorman, C M; Howard, B

1985-01-01

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure. Images PMID:3859838

Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line.

PubMed

Yoshino, T P; Wu, X J; Liu, H D

1998-09-01

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

PubMed Central

Lopata, M A; Cleveland, D W; Sollner-Webb, B

1984-01-01

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA. Images PMID:6589587

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation.

PubMed

Montoya, Misty M; Ansel, K Mark

2017-04-13

CD4 + T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4 + T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation

PubMed Central

Zhai, Hui-Hong; Meng, Juan; Wang, Jing-Bo; Liu, Zhen-Xiong; Li, Yuan-Fei; Feng, Shan-Shan

2014-01-01

AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. METHODS: The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. RESULTS: CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CONCLUSION: CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells. PMID:25110433

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation.

PubMed

Zhai, Hui-Hong; Meng, Juan; Wang, Jing-Bo; Liu, Zhen-Xiong; Li, Yuan-Fei; Feng, Shan-Shan

2014-08-07

To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

Identifying and targeting determinants of melanoma cellular invasion.

PubMed

Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L; Tan, BeeShin; Behren, Andreas; Cebon, Jonathan; McKeown, Sonja J

2016-07-05

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Identifying and targeting determinants of melanoma cellular invasion

PubMed Central

Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L.; Tan, BeeShin

2016-01-01

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients. PMID:27172792

Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents.

PubMed

Zhang, Guishui; Dass, Crispin R; Sumithran, Eric; Di Girolamo, Nick; Sun, Lun-Quan; Khachigian, Levon M

2004-05-05

The basic region-leucine zipper protein c-Jun has been linked to cell proliferation, transformation, and apoptosis. However, a direct role for c-Jun in angiogenesis has not been shown. We used human microvascular endothelial cells (HMEC-1) transfected with a DNAzyme targeting the c-Jun mRNA (Dz13), related oligonucleotides, or vehicle in in vitro models of microvascular endothelial cell proliferation, migration, chemoinvasion, and tubule formation, a rat model of corneal neovascularization, and a mouse model of solid tumor growth and vascular endothelial growth factor (VEGF)-induced angiogenesis. All statistical tests were two-sided. Compared with mock-transfected cells, HMEC-1 cells transfected with Dz13 expressed less c-Jun protein and possessed lower DNA-binding activity. Dz13 blocked endothelial cell proliferation, migration, chemoinvasion, and tubule formation. Dz13 inhibited the endothelial cell expression and proteolytic activity of MMP-2, a c-Jun-dependent gene. Dz13 inhibited VEGF-induced neovascularization in the rat cornea compared with vehicle control (Dz13 versus vehicle: 4.0 neovessels versus 30.7 neovessels, difference = 26.7 neovessels; P =.004; area occupied by new blood vessels for Dz13 versus vehicle: 0.35 mm2 versus 1.52 mm2, difference = 1.17 mm2; P =.005) as well as solid melanoma growth in mice (Dz13 versus vehicle at 14 days: 108 mm3 versus 283 mm3, difference = 175 mm3; P =.006) with greatly reduced vascular density (Dz13 versus vehicle: 30% versus 100%, difference = 70%; P<.001). DNAzymes targeting c-Jun may have therapeutic potential as inhibitors of tumor angiogenesis and growth.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

PubMed

Saxena, Shikha; Desai, G S; Kumar, G Ravi; Sahoo, A P; Santra, Lakshman; Singh, Lakshya Veer

2015-05-01

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

HSP60, a protein downregulated by IGFBP7 in colorectal carcinoma

PubMed Central

2010-01-01

Background In our previous study, it was well defined that IGFBP7 was an important tumor suppressor gene in colorectal cancer (CRC). We aimed to uncover the downstream molecules responsible for IGFBP7's behaviour in this study. Methods Differentially expressed protein profiles between PcDNA3.1(IGFBP7)-transfected RKO cells and the empty vector transfected controls were generated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) identification. The selected differentially expressed protein induced by IGFBP7 was confirmed by western blot and ELISA. The biological behaviour of the protein was explored by cell growth assay and colony formation assay. Results Six unique proteins were found differentially expressed in PcDNA3.1(IGFBP7)-transfected RKO cells, including albumin (ALB), 60 kDa heat shock protein(HSP60), Actin cytoplasmic 1 or 2, pyruvate kinase muscle 2(PKM2), beta subunit of phenylalanyl-tRNA synthetase(FARSB) and hypothetical protein. The downregulation of HSP60 by IGFBP7 was confirmed by western blot and ELISA. Recombinant human HSP60 protein could increase the proliferation rate and the colony formation ability of PcDNA3.1(IGFBP7)-RKO cells. Conclusion HSP60 was an important downstream molecule of IGFBP7. The downregulation of HSP60 induced by IGFBP7 may be, at least in part, responsible for IGFBP7's tumor suppressive biological behaviour in CRC. PMID:20433702

T-kininogen can either induce or inhibit proliferation in Balb/c 3T3 fibroblasts, depending on the route of administration.

PubMed

Aravena, M; Pérez, C; Pérez, V; Acuña-Castillo, C; Gómez, C; Leiva-Salcedo, E; Nishimura, S; Sabaj, V; Walter, R; Sierra, F

2005-03-01

T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

2015-09-14

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

KIR3DL2 binds to HLA-B27 dimers and free heavy chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis

PubMed Central

Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

2013-01-01

1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC. PMID:23440420

Pressure-Mediated Oligonucleotide Transfection of Rat and Human Cardiovascular Tissues

NASA Astrophysics Data System (ADS)

Mann, Michael J.; Gibbons, Gary H.; Hutchinson, Howard; Poston, Robert S.; Hoyt, E. Grant; Robbins, Robert C.; Dzau, Victor J.

1999-05-01

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Carbon nanoparticles for gene transfection in eukaryotic cell lines.

PubMed

Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

2014-06-01

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. Copyright © 2014 Elsevier B.V. All rights reserved.

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes

PubMed Central

Khuu, Cuong; Jevnaker, Anne-Marthe; Bryne, Magne; Osmundsen, Harald

2014-01-01

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40–50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92—or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed—about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to “Cellular Growth and Proliferation” and “Cell Cycle.” Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants. PMID:25202322

Flow-through electroporation based on constant voltage for large-volume transfection of cells.

PubMed

Geng, Tao; Zhan, Yihong; Wang, Hsiang-Yu; Witting, Scott R; Cornetta, Kenneth G; Lu, Chang

2010-05-21

Genetic modification of cells is a critical step involved in many cell therapy and gene therapy protocols. In these applications, cell samples of large volume (10(8)-10(9)cells) are often processed for transfection. This poses new challenges for current transfection methods and practices. Here we present a novel flow-through electroporation method for delivery of genes into cells at high flow rates (up to approximately 20 mL/min) based on disposable microfluidic chips, a syringe pump, and a low-cost direct current (DC) power supply that provides a constant voltage. By eliminating pulse generators used in conventional electroporation, we dramatically lowered the cost of the apparatus and improved the stability and consistency of the electroporation field for long-time operation. We tested the delivery of pEFGP-C1 plasmids encoding enhanced green fluorescent protein into Chinese hamster ovary (CHO-K1) cells in the devices of various dimensions and geometries. Cells were mixed with plasmids and then flowed through a fluidic channel continuously while a constant voltage was established across the device. Together with the applied voltage, the geometry and dimensions of the fluidic channel determined the electrical parameters of the electroporation. With the optimal design, approximately 75% of the viable CHO cells were transfected after the procedure. We also generalize the guidelines for scaling up these flow-through electroporation devices. We envision that this technique will serve as a generic and low-cost tool for a variety of clinical applications requiring large volume of transfected cells. Copyright 2010 Elsevier B.V. All rights reserved.

Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

PubMed

Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P

2000-07-03

In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells.

PubMed

Batman, Gavin; Oliver, Anthony W; Zehbe, Ingeborg; Richard, Christina; Hampson, Lynne; Hampson, Ian N

2011-01-01

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

GROWTH OF HUMAN PANCREATIC CANCER IS INHIBITED BY DOWN-REGULATION OF GASTRIN GENE EXPRESSION

PubMed Central

Matters, Gail L.; Harms, John F.; McGovern, Christopher O.; Jayakumar, Calpurnia; Crepin, Keisha; Smith, Zachary P.; Nelson, Melissa C.; Stock, Heather; Fenn, Craig W.; Kaiser, James; Kester, Mark; Smith, Jill P.

2009-01-01

Objectives This study evaluated the effects of gastrin mRNA down-regulation on growth of human pancreatic cancer. Methods Gastrin expression was examined in human pancreatic cancer cell lines by RT-PCR and peptide expression was assessed by immunocytochemistry. Gastrin was down-regulated using either stable transfection of an antisense gastrin cDNA or one of three shRNA (short hairpin RNA) constructs. Tumor formation was evaluated following either subcutaneous or orthotopic injections into nude mice. The effect of nanoliposomes loaded with gastrin siRNA was tested in mice bearing pancreatic tumors. Results Stable transfection of gastrin antisense or shRNAs into BxPC-3 cells resulted in clones with >90% reduction in gastrin mRNA. Tumor growth rate and incidence of metastases in both wild type and transfected pancreatic cancer cells was directly proportional to the degrees of gastrin mRNA expression. Immunofluoresence analysis confirmed that gastrin peptide levels were decreased in antisense and shRNA tumors. Gastrin knockdown clones had lower Ki-67 and increased cleaved caspase-3 staining, consistent with known effects of gastrin on proliferation and apoptosis. Tumors in mice treated with gastrin siRNA were smaller than controls. Conclusions These results suggest that RNAi targeting of gastrin could serve as an effective treatment for pancreatic cancer. PMID:19465883

Adenovirus‑mediated overexpression of cystic fibrosis transmembrane conductance regulator enhances invasiveness and motility of serous ovarian cancer cells.

PubMed

Xu, Jiao; Lin, Liangbo; Yong, Min; Dong, Xiaojing; Yu, Tinghe; Hu, Lina

2016-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the adenosine triphosphate‑binding cassette transporter family, members of which are involved in several types of cancer. Previous studies by our group reported that CFTR was highly expressed in serous ovarian cancer (SOC) tissues, and that knockdown of CFTR suppressed the proliferation of ovarian cancer in vitro and in vivo. Thus, the aim of the present study was to construct a recombinant adenoviral vector for the expression of the human CFTR gene in order to study the role of CFTR overexpression in the malignant invasion and migration of SOC cells in vitro. The present study then focused on the mechanisms of the role of CFTR in the migratory and invasive malignant properties of SOC cells. The CFTR gene was inserted into an adenoviral vector by using the AdEasy system in order to obtain the Ad‑CFTR overexpression vector, which was used to transfect the A2780 SOC cell line. Reverse-transcription polymerase chain reaction, western blot analysis and immunofluorescence were performed to detect the expression and localization of CFTR. Cell invasion and motility of the transfected cells compared with those of control cells were observed using Transwell and wound healing assays. A ~4,700 bp fragment of the CFTR gene was confirmed to be correctly cloned in the adenoviral vector and amplification of Ad‑CFTR was observed in HEK293 cells during package. After 48 h of transfection with Ad‑CFTR, ~90% of A2780 cells were red fluorescence protein‑positive. Immunofluorescence showed that following transfection, CFTR expression was increased and CFTR was located in the cell membrane and cytoplasm. CFTR overexpression was shown to enhance the invasion and motility of A2780 cells in vitro. Furthermore, the effects of CFTR overexpression on the activation c‑Src signaling were observed by western blot analysis. CFTR overexpressing cells showed the lowest activity of phospho‑Src (Tyr530), suggesting that CFTR may affect the activation of c‑Src signaling. The results of the present study demonstrated that adenovirus‑mediated CFTR overexpression enhanced cell invasion and motility of SOC cells in vitro. Furthermore, CFTR may be critical for the activation of c‑Src signaling.

Untying a nanoscale knotted polymer structure to linear chains for efficient gene delivery in vitro and to the brain

NASA Astrophysics Data System (ADS)

Newland, B.; Aied, A.; Pinoncely, A. V.; Zheng, Y.; Zhao, T.; Zhang, H.; Niemeier, R.; Dowd, E.; Pandit, A.; Wang, W.

2014-06-01

The purpose of this study was to develop a platform transfection technology, for applications in the brain, which could transfect astrocytes without requiring cell specific functionalization and without the common cause of toxicity through high charge density. Here we show that a simple and scalable preparation technique can be used to produce a ``knot'' structured cationic polymer, where single growing chains can crosslink together via disulphide intramolecular crosslinks (internal cyclizations). This well-defined knot structure can thus ``untie'' under reducing conditions, showing a more favorable transfection profile for astrocytes compared to 25 kDa-PEI (48-fold), SuperFect® (39-fold) and Lipofectamine®2000 (18-fold) whilst maintaining neural cell viability at over 80% after four days of culture. The high transfection/lack of toxicity of this knot structured polymer in vitro, combined with its ability to mediate luciferase transgene expression in the adult rat brain, demonstrates its use as a platform transfection technology which should be investigated further for neurodegenerative disease therapies.The purpose of this study was to develop a platform transfection technology, for applications in the brain, which could transfect astrocytes without requiring cell specific functionalization and without the common cause of toxicity through high charge density. Here we show that a simple and scalable preparation technique can be used to produce a ``knot'' structured cationic polymer, where single growing chains can crosslink together via disulphide intramolecular crosslinks (internal cyclizations). This well-defined knot structure can thus ``untie'' under reducing conditions, showing a more favorable transfection profile for astrocytes compared to 25 kDa-PEI (48-fold), SuperFect® (39-fold) and Lipofectamine®2000 (18-fold) whilst maintaining neural cell viability at over 80% after four days of culture. The high transfection/lack of toxicity of this knot structured polymer in vitro, combined with its ability to mediate luciferase transgene expression in the adult rat brain, demonstrates its use as a platform transfection technology which should be investigated further for neurodegenerative disease therapies. Electronic supplementary information (ESI) available: 1H NMR spectroscopy data and gel permeation chromatography data. See DOI: 10.1039/c3nr06737h

Selective Inhibition of the Human tie-1 Promoter with Triplex-Forming Oligonucleotides Targeted to Ets Binding Sites

PubMed Central

Hewett, Peter W; Daft, Emma L; Laughton, Charles A; Ahmad, Shakil; Ahmed, Asif; Murray, J Clifford

2006-01-01

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21–22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (Kd ~10−7 M) at 37 °C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction. PMID:16838069

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites.

PubMed

Hewett, Peter W; Daft, Emma L; Laughton, Charles A; Ahmad, Shakil; Ahmed, Asif; Murray, J Clifford

2006-01-01

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

PubMed

Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

2016-05-27

The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

PubMed

Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

2010-01-01

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

Enhanced phosphorylation of STAT1 is dependent on PKR signaling in HLA-B27 expressing U937 monocytic cells

PubMed Central

Ruuska, Marja; Sahlberg, Anna S.; Colbert, Robert A.; Granfors, Kaisa; Penttinen, Markus A.

2011-01-01

Objective To study the phosphorylation of STAT1 in HLA-B27-transfected human monocytic cells and the role of signaling molecules PKR and p38 in STAT1 phosphorylation. Methods U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains (HC) with amino acid substitutions in the B pocket were prepared. Mock transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. PMA differentiated cells were stimulated with LPS or infected with S. enteritidis. Western blotting and flow cytometry were used to detect the phosphorylation and expression levels of STAT1 protein. Specific inhibitors were added in cell culture to study the role of PKR and p38 on STAT1 phosphorylation. Results STAT1 is constitutively highly phosphorylated on tyrosine 701 residue in HLA-B27 positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype is associated with the expression of HLA-B27 HCs that misfold. In addition, phosphorylation of STAT1 is dependent on PKR. Conclusion Our results show that STAT1 tyrosine 701 is constitutively highly phosphorylated in HLA-B27 expressing monocyte-macrophage cell line. Since phosphorylation of tyrosine 701 on STAT1 is sufficient to induce interferon-dependent genes, constitutive activity of this phosphorylation site may lead to overexpression of interferon-dependent genes, as well as other STAT1-dependent genes, in HLA-B27 monocyte-macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT1, a critical regulator of the inflammatory response. PMID:21968657

[Efficacy of siRNA on feline leukemia virus replication in vitro].

PubMed

Lehmann, Melanie; Weber, Karin; Rauch, Gisep; Hofmann-Lehmann, Regina; Hosie, Margaret J; Meli, Marina L; Hartmann, Katrin

2015-01-01

Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 μg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition.

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

The Expression of BTS-2 Enhances Cell Growth and Invasiveness in Renal Cell Carcinoma.

PubMed

Pham, Quoc Thang; Oue, Naohide; Yamamoto, Yuji; Shigematsu, Yoshinori; Sekino, Yohei; Sakamoto, Naoya; Sentani, Kazuhiro; Uraoka, Naohiro; Tiwari, Mamata; Yasui, Wataru

2017-06-01

Renal cell carcinoma (RCC) is one of the most common types of cancer in developed countries. Bone marrow stromal cell antigen 2 (BST2) gene, which encodes BST2 transmembrane glycoprotein, is overexpressed in several cancer types. In the present study, we analyzed the expression and function of BST2 in RCC. BST2 expression was analyzed by immunohistochemistry in 123 RCC cases. RNA interference was used to inhibit BST2 expression in a RCC cell line. Immunohistochemical analysis showed that 32% of the 123 RCC cases were positive for BST2. BST2 expression was positively associated with tumour stage. Furthermore, BST2 expression was an independent predictor of survival in patients with RCC. BST2 siRNA-transfected Caki-1 cells displayed significantly reduced cell growth and invasive activity relative to negative control siRNA-transfected cells. These results suggest that BST2 plays an important role in the progression of RCC. Because BST2 is expressed on the cell membrane, BST2 is a good therapeutic target for RCC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

GAP JUNCTION COMMUNICATON IN A TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS

EPA Science Inventory

GAP JUNCTION COMMUNICTION IN TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS.

OBJECTIVE: We previously showed that functional gap junction communication (GJC), as monitored by dye transfer (DT), could be enhanced in mouse C3H 10T112 cells and in mouse...

Podoplanin increases migration and angiogenesis in malignant glioma

PubMed Central

Grau, Stefan J; Trillsch, Fabian; Tonn, Joerg-Christian; Goldbrunner, Roland H; Noessner, Elfriede; Nelson, Peter J; von Luettichau, Irene

2015-01-01

Expression of podoplanin in glial brain tumors is grade dependent. While serving as a marker for tumor progression and modulating invasion in various neoplasms, little is known about podoplanin function in gliomas. Therefore we stably transfected two human glioma cell lines (U373MG and U87MG) with expression plasmids encoding podoplanin. The efficacy of transfection was confirmed by FACS analysis, PCR and immunocytochemistry. Cells were then sorted for highly podoplanin expressing cells (U373Phigh/U87Phigh). Transfection did not influence the production of pro-angiogenic factors including VEGF, VEGF-C and D. Also, expression of VEGF receptors (VEGFR) remained unchanged except for U87Phigh, where a VEGFR3 expression was induced. U373Phigh showed significantly reduced proliferation as compared to mock transfected group. By contrast, podoplanin significantly increased migration and invasion into collagen matrix. Furthermore, conditioned media from Phigh glioma cells strongly induced tube formation on matrigel. In conclusion, podoplanin increased migration of tumor cells and enhanced tube formation activity in endothelial cells independent from VEGF. Thus, podoplanin expression may be an important step in tumor progression. PMID:26339454

MicroRNA-142-5p Overexpression Inhibits Cell Growth and Induces Apoptosis by Regulating FOXO in Hepatocellular Carcinoma Cells.

PubMed

Lou, Kexin; Chen, Ning; Li, Zhihong; Zhang, Bei; Wang, Xiuli; Chen, Ye; Xu, Haining; Wang, Dongwei; Wang, Hao

2017-01-02

Abnormal expression of microRNA (miR)-142-5p has been reported in hepatocellular carcinoma (HCC). However, little information is available regarding the functional role of miR-142-5p in HCC. We aimed to explore the effects of miR-142-5p aberrant expression on HCC cell growth and cell apoptosis, as well as the underlying mechanism. Human HCC cell lines HepG2 and SMMC-7721 cells were transfected with miR-142-5p mimic, inhibitor, or a corresponding negative control. Cell viability, cell cycle distribution, and cell apoptosis were then analyzed. In addition, protein expression of Forkhead box, class O (FOXO) 1 and 3, a Bcl-2-interacting mediator of cell death (Bim), procaspase 3, and activated caspase 3 was measured. After transfection with miR-142-5p inhibitor, FOXO1 and FOXO3 were overexpressed, and then the cell viability and cell apoptosis were determined again. The relative cell viability in both HepG2 and SMMC-7721 cells was significantly reduced by miR-142-5p overexpression (p < 0.05). miR-142-5p overexpression displayed a significant blockage at the G1/S transition and significantly increased the percentages of G0/G1 phase. Moreover, the results showed that miR-142-5p overexpression significantly induced cell apoptosis and statistically elevated the protein expression levels of FOXO1, FOXO3, Bim, procaspase 3, and activated caspase 3. However, the cells transfected with miR-142-5p inhibitor showed contrary results. Additionally, the effects of miR-142-5p inhibitor on cell viability and apoptosis were reversed by overexpression of FOXO. In conclusion, our results suggest that miR-142-5p overexpression shows an important protective role in HCC by inhibiting cell growth and inducing apoptosis. These effects might be by regulating FOXO expression in HCC cells.

Regulation of D-cyclin translation inhibition in myeloma cells treated with mTOR inhibitors: Rationale for combined treatment with ERK inhibitors and rapamycin

PubMed Central

Frost, Patrick; Shi, Yijiang; Hoang, Bao; Gera, Joseph; Lichtenstein, Alan

2009-01-01

We have shown that heightened AKT activity sensitized multiple myeloma (MM) cells to the anti-tumor effects of the mTOR-inhibitor, CCI-779. To test the mechanism of AKT’s regulatory role, we stably transfected U266 MM cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analog, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mTOR inhibitors to downregulate D-cyclin expression was significantly greater in AKT-transfected MM cells, due in part, to AKT’s ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was demonstrated in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild type PTEN. As ERK/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells demonstrated significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. However, ectopic over-expression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high AKT” myeloma cells indicating that MEK/ERK activity was required but not sufficient for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in MM cells and ERK facilitates activity. PMID:19139116

Simulation of micro/nano electroporation for cell transfection

NASA Astrophysics Data System (ADS)

Zhang, Guocheng; Fan, Na; Jiang, Hai; Guo, Jian; Peng, Bei

2018-03-01

The 3D micro/nano electroporation for transfection has become a powerful biological cell research technique with the development of micro-nano manufacturing technology. The micro channels connected the cells with transfection reagents on the chip were important to the transmemnbrane potentical, which directly influences the electroporation efficiency. In this study, a two-dimensional model for electroporation of cells was designed to address the effects of channels’ sizes and number on transmembrane potential. The simulation results indicated that the transmembrane potential increased with increasing size of channels’ entrances. Moreover, compared with single channel entrance, the transmembrane potential was higher when the cells located at multiple channels entrances. These results suggest that it IS required to develop higher micro manufacturing technology to create channels as we expected size.

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury.

PubMed

Dong, Yuzhen; Yang, Libin; Yang, Lin; Zhao, Hongxing; Zhang, Chao; Wu, Dapeng

2014-08-15

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesenchymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.

PubMed

Oem, Jae-Ku; Xiang, Zhonghua; Zhou, Yan; Babiuk, Lorne A; Liu, Qiang

2007-09-01

Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis. To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation. A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection. Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity. Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.

Measurement of DNA repair deficiency in workers exposed to benzene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hallberg, L.M.; Au, W.W.; El Zein, R.

1996-05-01

We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m{sup 2} UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repairedmore » normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m{sup 2} and 350 J/m{sup 2} were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (<0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. 26 refs., 4 figs., 2 tabs.« less

The influence of femtosecond laser pulse wavelength on embryonic stem cell differentiation

NASA Astrophysics Data System (ADS)

Mthunzi, Patience

2012-10-01

Stem cells are rich in proteins, carbohydrates, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and various other cellular components which are responsible for a diversity of functions. Mostly the building blocks of these intracellular entities play an active role in absorbing ultra-violet (UV) and visible light sources. Light-matter interactions in biomaterials are a complex situation and subsequent damage may not always amount only from wavelength dependent effects but may also be driven by a wealth of other optical parameters which may lead to a variety photochemical reactions. Previously, literature has reported efficient photo-transfection and differentiation of pluripotent stem cells via near infrared (NIR) femtosecond (fs) laser pulses with minimum compromise to their viability. Therefore, in this study the influence of using different fs laser wavelengths on optical stem cell transfection and differentiation is investigated. A potassium titanyl phosphate (KTP) crystal was employed in frequency doubling a 1064 nm fs laser beam. The newly generated 532 nm fs pulsed beam was then utilized for the first time in transient photo-transfection of ES-E14TG2a mouse embryonic stem (mES) cells. Compared to using 1064 nm fs pulses which non-invasively introduce plasmid DNA and other macromolecules into mES cells, our results showed a significant decline in the photo-transfection efficiency following transfecting with a pulsed fs visible green beam.

KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis.

PubMed

Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

2013-04-01

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical β2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

Glycogen synthase kinase-3beta and the p25 activator of cyclin dependent kinase 5 increase pausing of mitochondria in neurons.

PubMed

Morel, M; Authelet, M; Dedecker, R; Brion, J P

2010-06-02

The complex bi-directional axoplasmic transport of mitochondria is essential for proper metabolic functioning of neurons and is controlled by phosphorylation. We have investigated by time-lapse imaging the effects of increased expression of glycogen synthase kinase-3beta (GSK-3beta) and of the p25 activator of cyclin dependent kinase 5 on mitochondria movements in mammalian cortical neurons and in PC12 cells. Both GSK-3beta and p25 increased the stationary behaviour of mitochondria in PC12 and in neurons, decreased their anterograde transport but did not affect the intrinsic velocities of mitochondria. The microtubule-associated tau proteins were more phosphorylated in GSK-3beta and p25 transfected neurons, but ultrastructural observation showed that these cells still contained microtubules and nocodazole treatment further reduced residual mitochondria movements in GSK-3beta or p25 transfected neurons, indicating that microtubule disruption was not the primary cause of increased mitochondrial stationary behaviour in GSK-3beta or p25 transfected neurons. Our results suggest that increased expression of GSK-3beta and p25 acted rather by decreasing the frequency of mitochondrial movements driven by molecular motors and that GSK-3beta and p25 might regulate these transports by controlling the time that mitochondria spend pausing, rather than their velocities. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

PubMed

Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

2009-06-01

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine.

PubMed

Schomaker, Markus; Heinemann, Dag; Kalies, Stefan; Willenbrock, Saskia; Wagner, Siegfried; Nolte, Ingo; Ripken, Tammo; Murua Escobar, Hugo; Meyer, Heiko; Heisterkamp, Alexander

2015-02-03

In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Nonviral transfection of suspension cells in ultrasound standing wave fields.

PubMed

Lee, Yu-Hsiang; Peng, Ching-An

2007-05-01

Ultrasound-induced cavitation has been widely used for delivering DNA vectors into cells. However, this approach may seriously disrupt cell membranes and cause lethal damage when cells are exposed to the inertial cavitation field. In this study, instead of using sonoporation, ultrasound standing wave fields (USWF) were explored for nonviral transfection of suspension cells. Acoustic resonance in a tubular chamber was generated from the interference of waves emitted from a piezoelectric transducer and consequently reflected from a borosilicate glass coverslip. The suspended K562 erythroleukemia cells were transfected by polyethyleneimine (PEI)/DNA complexes with and without exposure to 1-MHz USWF for 5 min. During USWF exposure, K562 cells moved to the pressure nodal planes first and formed cell bands by the primary radiation force. Nanometer-sized PEI/DNA complexes, circulated between nodal planes by acoustic microstreaming, then used the cell agglomerates as the nucleating sites on which to attach. After incubation at 37 degrees C for 48 h, the efficiency of nonviral transfection based on EGFP transgene expression was determined by fluorescent microscopy and fluorometry. Both studies showed that USWF brought suspended K562 cells and PEI/DNA complexes into close contact at the pressure nodal planes, yielding an approximately 10-fold increment of EGFP transgene expression compared with the group without ultrasonic treatment.

Journal of Virology

Science.gov Websites

Herpes Simplex Virus 1 Improved Control of Simian/Human Immunodeficiency Virus in Macaques following Hemisphere Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human -associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. Temporal

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-I/sup n/ (NIH-3T3) and two Fv-I/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 genotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-1 cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells.

PubMed

Mennesson, Eric; Erbacher, Patrick; Piller, Véronique; Kieda, Claudine; Midoux, Patrick; Pichon, Chantal

2005-06-01

Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes. Copyright (c) 2005 John Wiley & Sons, Ltd.

HLA-B27 Modulates Intracellular Growth of Salmonella Pathogenicity Island 2 Mutants and Production of Cytokines in Infected Monocytic U937 Cells

PubMed Central

Ge, Shichao; He, Qiushui; Granfors, Kaisa

2012-01-01

Background Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells. Methods/Principal Findings To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis. Conclusions The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis. PMID:22470519

Heme oxygenase-1 protects INF-gamma primed endothelial cells from Jurkat T-cell adhesion.

PubMed

Du, D; Chang, S; Chen, B; Zhou, H; Chen, Z K

2007-12-01

The heme oxygenase-1 (HO-1) system is associated with the rate-limiting step of conversion of heme, one of the most critical roles in cytoprotective mechanisms. Our study investigated its potential role in protection of endothelial cells from T cells. The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells. Indirect fluorescent staining was used to examine the expression of HO-1 protein. Then endothelial cells primed by INF-gamma were mixed in culture with Jurkat T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The number of adhesive Jurkat T cells was determined using FACS to evaluate the adhesion effect. After being cultured with endothelial cells, the cell cycle of Jurkat T cells was detected using FACS. Expression of HO-1 on endothelial cells conferred significant protection against Jurkat T-cell-mediated adhesion. The rate of Jurkat T-cell adhesions was reduced to 19.06%, in contrast with 31.42% in the control group (P<.05). After using ZnPP, an inhibitor of HO-1, the rate of Jurkat T-cell adhesion recovered to 29.08%. The binding activities between endothelial cells and Jurkat T cells was blocked by HO-1 expression. The proliferation of Jurkat T cells was inhibited after culture with endothelial cells, which had been transfected with HO-1, which blocked cell cycle entry of T cells. More than 60% of Jurkat T cells remained in G0/G1 compared with 40% among the control group. HO-1 directly protected endothelial cells primed by INF-gamma from Jurkat T cells and down-regulated the expression of HLA-DR on the surface of endothelial cells. These results indicated that transgenic expression of HO-1 may be useful to prevent lymphocytes from responding to endothelial cells.

Role of caspase-9 in the effector caspases and genome expressions, and growth of bovine skeletal myoblasts.

PubMed

Van Ba, Hoa; Hwang, Inho

2014-02-01

Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48 h. Results indicated that all three siRNAs could silence the caspase-9 mRNA expression significantly. Particularly, the mRNA expression level of caspase-9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase-9 led to the significant increases in the mRNA and protein expressions of effector caspase-3, whereas the reduction in mRNA and protein expressions of caspase-7. The microarray results showed that the suppression of caspase-9 resulted in significant upregulations of cell proliferation-, adhesion-, growth-, development- and division-regulating genes, whereas the reduction in the expressions of cell death program- and stress response-regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase-9 could play an important role in the control of cell growth, and knockdown of caspase-9 may have genuine potential in the treatment of skeletal muscle atrophy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Aspects of nonviral gene therapy: correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids.

PubMed

Parvizi, Paria; Jubeli, Emile; Raju, Liji; Khalique, Nada Abdul; Almeer, Ahmed; Allam, Hebatalla; Manaa, Maryem Al; Larsen, Helge; Nicholson, David; Pungente, Michael D; Fyles, Thomas M

2014-01-30

This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

NASA Astrophysics Data System (ADS)

Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-11-01

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors.

PubMed

Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-11-17

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.