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Sample records for rna transfected autologous

  1. Antibody production by in vivo RNA transfection.

    PubMed

    Romani, Bizhan; Kavyanifard, Amirarsalan; Allahbakhshi, Elham

    2017-09-07

    Monoclonal antibodies have a variety of applications in research and medicine. Here, we report development of a new method for production of monoclonal antibodies. Our method relies on in vivo RNA transfection rather than peptide vaccination. We took advantage of RNA transcripts complexed with DOTMA and DOPE lipids to transfect mice. Intravenous administration of our RNA vaccine to mice resulted in expression of the antigenic peptides by splenic dendritic cells and detection of the antigens in the serum. The RNA vaccine stimulated production of specific antibodies against the RNA-encoded peptides. We produced monoclonal antibodies against viral, bacterial, and human antigens. In addition, we showed that our RNA vaccine stimulated humoral immunity and rescued mice infected with influenza A virus. Our method could be used as an efficient tool to generate monoclonal antibodies and to stimulate humoral immunity for research and medical purposes.

  2. Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

    PubMed Central

    Angel, Matthew; Yanik, Mehmet Fatih

    2010-01-01

    Background Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death. Methodology/Principal Findings RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-β (Ifnb1), Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division. Conclusions/Significance Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection. PMID:20668695

  3. A General RNA Motif for Cellular Transfection

    PubMed Central

    Magalhães, Maria LB; Byrom, Michelle; Yan, Amy; Kelly, Linsley; Li, Na; Furtado, Raquel; Palliser, Deborah; Ellington, Andrew D; Levy, Matthew

    2012-01-01

    We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells. PMID:22233578

  4. [Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells].

    PubMed

    Li, Ji; Liu, Zuojin

    2015-12-01

    To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells. Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (P<0.05). In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.

  5. Human breast adipose-derived stem cells transfected with the stromal cell-derived factor-1 receptor CXCR4 exhibit enhanced viability in human autologous free fat grafts.

    PubMed

    Xu, Fang-tian; Li, Hong-mian; Yin, Qing-Shui; Liu, Da-lie; Nan, Hua; Zhao, Pei-ran; Liang, Shuang-wu

    2014-01-01

    The main complication of autologous free fat tissue transplantation is fat resorption and calcification due to the ischemic necrosis of fat. The promotion of transplant neovascularization soon after autologous free fat grafts may reduce these outcomes. In adulthood, stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. We hypothesized that CXCR4 may improve the long-term survival of free fat tissue transplants by recruiting endothelial progenitor cells (EPCs) and may therefore improve graft revascularization. In this study, we aimed to determine the effect of human breast adipose-derived stem cells (HBASCs) transfected with the CXCR4 gene on the survival rate of human autologous free fat transplants in nude mice. Human breast adipose-derived stem cells (HBASCs) were expanded ex vivo for 3 passages, labeled with green fluorescent protein (GFP) and transfected with CXCR4 or left untransfected. Autologous fat tissues were mixed with the GFP-labeled, CXCR4-transfected HBASCs (group A), GFP-labeled HBASCs (group B), the known vascularization-promoting agent VEGF (group C), or medium (group D) and then injected subcutaneously into 32 nude mice at 4 spots in a random fashion. Six months later, the transplanted tissue volume and histology were evaluated, and neo-vascularization was quantified by counting the capillaries. CXCR4 and SDF-1α mRNA expression in the transplants was determined using real-time quantitative PCR analysis (qPCR). The data revealed that the control (group D) transplant volume survival was 28.3 ± 4.5%. Mixing CXCR4-transfected (group A) and untransfected (group B) HBASCs significantly increased transplant volume survival (79.5 ± 8.3% and 67.2 ± 5.9%, respectively), whereas VEGF-transfected HBASCs (group C) were less effective (41.2 ± 5.1%). Histological analysis revealed that both types

  6. Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of patients with malignant gliomas

    PubMed Central

    Okada, Hideho; Lieberman, Frank S; Walter, Kevin A; Lunsford, L Dade; Kondziolka, Douglas S; Bejjani, Ghassan K; Hamilton, Ronald L; Torres-Trejo, Alejandro; Kalinski, Pawel; Cai, Quan; Mabold, Jennifer L; Edington, Howard D; Butterfield, Lisa H; Whiteside, Theresa L; Potter, Douglas M; Schold, S Clifford; Pollack, Ian F

    2007-01-01

    Background The prognosis for malignant gliomas remains dismal. We addressed the safety, feasibility and preliminary clinical activity of the vaccinations using autologous glioma cells and interleukin (IL)-4 gene transfected fibroblasts. Methods In University of Pittsburgh Cancer Institute (UPCI) protocol 95-033, adult participants with recurrent glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) received gross total resection (GTR) of the recurrent tumors, followed by two vaccinations with autologous fibroblasts retrovirally transfected with TFG-IL4-Neo-TK vector admixed with irradiated autologous glioma cells. In UPCI 99-111, adult participants with newly diagnosed GBM or AA, following GTR and radiation therapy, received two intradermal vaccinations with the TFG-IL4-Neo-TK-transfected fibroblasts admixed with type-1 dendritic cells (DC) loaded with autologous tumor lysate. The participants were evaluated for occurrence of adverse events, immune response, and clinical response by radiological imaging. Results and Discussion In UPCI 95-033, only 2 of 6 participants received the vaccinations. Four other participants were withdrawn from the trial because of tumor progression prior to production of the cellular vaccine. However, both participants who received two vaccinations demonstrated encouraging immunological and clinical responses. Biopsies from the local vaccine sites from one participant displayed IL-4 dose-dependent infiltration of CD4+ as well as CD8+ T cells. Interferon (IFN)-γ Enzyme-Linked Immuno-SPOT (ELISPOT) assay in another human leukocyte antigen (HLA)-A2+ participant demonstrated systemic T-cell responses against an HLA-A2-restricted glioma-associated antigen (GAA) epitope EphA2883–891. Moreover, both participants demonstrated clinical and radiological improvement with no evidence of allergic encephalitis, although both participants eventually succumbed with the tumor recurrence. In 99-111, 5 of 6 enrolled participants received scheduled

  7. Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

    PubMed Central

    Collis, P S; O'Donnell, B J; Barton, D J; Rogers, J A; Flanegan, J B

    1992-01-01

    Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis. Images PMID

  8. Construction of a Hep-2 cell line stably transfected with Livin shRNA.

    PubMed

    Wang, S L; Deng, W T; Wen, G F; Li, C W; Zeng, Y J

    2016-01-01

    The aim of this study was to construct a eukaryotic expression plasmid with a short hairpin RNA (shRNA) targeting Livin in order to obtain a stably transfected Hep-2 cell line with a reduced expression of Livin. The shRNA targeting Livin mRNA was designed, and a shRNA plasmid and a negative control plasmid were constructed. After amplification in E. coli, restriction endonuclease digestion and sequence confirmation, the plasmids were transfected into Hep-2 cells using Lipofectamine 2000. The stably transfected cell line was screened using G418, and inhibition of Livin mRNA and protein levels were detected using real-time PCR and western blotting, respectively. pGenesil-Livin-shRNA eukaryotic expression plasmid was successfully constructed and identified by sequencing. Green fluorescent protein (GFP) expression was observed in Hep-2 cells transfected with shRNA plasmids by fluorescence microscopy. The expression levels of Livin mRNA and protein decreased significantly in Hep-2 cells transfected with the shRNA recombinant plasmid. The mRNA level was reduced by 47.17 %, and the protein level was reduced by 34.25 %. The shRNA eukaryotic expression plasmid targeting Livin was successfully constructed, which could significantly inhibit the expression of Livin in laryngeal cancer Hep-2 cells. This provides a basis for future research on the function of Livin in Hep-2 cells, and gene therapy for laryngeal cancer.

  9. Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format.

    PubMed

    Phua, Kyle K L; Leong, Kam W; Nair, Smita K

    2013-03-28

    Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

  10. Transfection of microRNA Mimics Should Be Used with Caution

    PubMed Central

    Jin, Hyun Yong; Gonzalez-Martin, Alicia; Miletic, Ana V.; Lai, Maoyi; Knight, Sarah; Sabouri-Ghomi, Mohsen; Head, Steven R.; Macauley, Matthew S.; Rickert, Robert C.; Xiao, Changchun

    2015-01-01

    Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics. PMID:26697058

  11. Molecular determinants for cyclo-oligosaccharide-based nanoparticle-mediated effective siRNA transfection.

    PubMed

    Manzanares, Darío; Araya-Durán, Ingrid; Gallego-Yerga, Laura; Játiva, Pablo; Márquez-Miranda, Valeria; Canan, Jonathan; Jiménez Blanco, José Luis; Mellet, Carmen Ortiz; González-Nilo, Fernando Danilo; García Fernández, José Manuel; Ceña, Valentín

    2017-07-01

    To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector. siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics. The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA. The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.

  12. Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages

    PubMed Central

    Ng, Yee Seng; Roca, Hernan; Fuller, David; Sud, Sudha

    2012-01-01

    Background MicroRNAs (miRNAs) are short non-coding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Results Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Conclusions Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best

  13. mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells.

    PubMed

    De Temmerman, Marie-Luce; Dewitte, Heleen; Vandenbroucke, Roosmarijn E; Lucas, Bart; Libert, Claude; Demeester, Jo; De Smedt, Stefaan C; Lentacker, Ine; Rejman, Joanna

    2011-12-01

    In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation

    PubMed Central

    Zhao, Yangbing; Zheng, Zhili; Cohen, Cyrille J.; Gattinoni, Luca; Palmer, Douglas C.; Restifo, Nicholas P.; Rosenberg, Steven A.; Morgan, Richard A.

    2006-01-01

    The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes. PMID:16140584

  15. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes.

  16. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  17. 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

  18. Mechanisms of Nanoparticle Mediated siRNA Transfection by Melittin-Derived Peptides

    PubMed Central

    Hou, Kirk K.; Pan, Hua; Ratner, Lee; Schlesinger, Paul H.; Wickline, Samuel A.

    2014-01-01

    Traditional peptide-mediated siRNA transfection via peptide transduction domains exhibits limited cytoplasmic delivery of siRNA due to endosomal entrapment. This work overcomes these limitations with the use of membrane-destabilizing peptides derived from melittin for the knockdown of NFkB signaling in a model of adult T-Cell leukemia/lymphoma. While the mechanism of siRNA delivery into the cytoplasmic compartment by peptide transduction domains has not been well studied, our analysis of melittin derivatives indicates that concurrent nanocomplex disassembly and peptide-mediated endosomolysis are crucial to siRNA transfection. Importantly, in the case of the most active derivative, p5RHH, this process is initiated by acidic pH, indicating that endosomal acidification after macropinocytosis can trigger siRNA release into the cytoplasm. These data provide general principles regarding nanocomplex response to endocytosis which may guide the development of peptide/siRNA nanocomplex-based transfection. PMID:24053333

  19. Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection

    PubMed Central

    Sheikhi Mehrabadi, Fatemeh; Zeng, Hanxiang; Johnson, Mark; Schlesener, Cathleen

    2015-01-01

    Summary The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape. PMID:26124878

  20. Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons

    PubMed Central

    Tönges, Lars; Lingor, Paul; Egle, Roman; Dietz, Gunnar P.H.; Fahr, Alfred; Bähr, Mathias

    2006-01-01

    RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents. PMID:16699166

  1. Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection.

    PubMed

    Uchida, Hirokuni; Itaka, Keiji; Nomoto, Takahiro; Ishii, Takehiko; Suma, Tomoya; Ikegami, Masaru; Miyata, Kanjiro; Oba, Makoto; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2014-09-03

    Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

  2. Efficient rescue of foot-and-mouth disease virus in cultured cells transfected with RNA extracted from clinical samples.

    PubMed

    Bisht, Punam; Mohapatra, Jajati K; Subramaniam, Saravanan; Das, Biswajit; Pande, Veena; Biswal, Jitendra K; Sharma, Gaurav K; Rout, Manoranjan; Ranjan, Rajeev; Dash, Bana B; Sanyal, Aniket; Pattnaik, Bramhadev

    2014-02-01

    In this study, an RNA transfection was used to rescue infectious foot-and-mouth disease (FMD) virus from clinical samples in BHK-21 cell line for diagnosis of FMD. Tissue samples (n=190) were subjected to FMD virus isolation by conventional cell culture and also by RNA transfection. FMD virus was isolated from 62% of the clinical samples by RNA transfection, whereas virus was isolated only from 16% of the clinical samples in conventional cell culture method, suggesting better performance of the RNA transfection. Virus was rescued from 67% and 10% of ELISA negative but multiplex PCR positive samples by RNA transfection and conventional cell culture, respectively. The efficiency of transfection was studied on clinical samples subjected to temperature as high as 37°C and varying pH (pH 4-9). Except up to 1 week of storage at 4°C at pH 7.5, virus isolation was not possible by cell culture. Virus was rescued by transfection from samples stored at 4°C for any of the applied pH up to 4 weeks, and when stored at 37°C virus could be rescued up to 4 weeks at pH 7.5 suggesting the fitness of transfection to isolate virus from clinical samples stored under inappropriate conditions. The sequence data and antigenic relationships with the vaccine strains, between virus rescued by transfection and conventional cell culture, were comparable. The RNA transfection will help to increase the efficiency of virus isolation, diagnosis and molecular epidemiological studies.

  3. Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

    PubMed Central

    Pepin, M C; Barden, N

    1991-01-01

    Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids. Images PMID:1996114

  4. High efficiency, site-specific transfection of adherent cells with siRNA using microelectrode arrays (MEA).

    PubMed

    Patel, Chetan; Muthuswamy, Jit

    2012-09-13

    The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region

  5. Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis

    PubMed Central

    Ho, Nicholas R. Y.; Usmani, Abul R.; Yin, Yan; Ma, Liang; Conrad, Donald F.

    2016-01-01

    Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6–9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for

  6. Efficient Protection and Transfection of Small Interfering RNA by Cationic Shell-Crosslinked Knedel-Like Nanoparticles

    PubMed Central

    Shen, Yuefei; Fang, Huafeng; Zhang, Ke; Shrestha, Ritu; Wooley, Karen L.

    2013-01-01

    Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake. PMID:23557117

  7. Discovery of siRNA lipid nanoparticles to transfect suspension leukemia cells and provide in vivo delivery capability.

    PubMed

    He, Wei; Bennett, Michael J; Luistro, Leopoldo; Carvajal, Daisy; Nevins, Thomas; Smith, Melissa; Tyagi, Gaurav; Cai, James; Wei, Xin; Lin, Tai-An; Heimbrook, David C; Packman, Kathryn; Boylan, John F

    2014-02-01

    As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

  8. Discovery of siRNA Lipid Nanoparticles to Transfect Suspension Leukemia Cells and Provide In Vivo Delivery Capability

    PubMed Central

    He, Wei; Bennett, Michael J; Luistro, Leopoldo; Carvajal, Daisy; Nevins, Thomas; Smith, Melissa; Tyagi, Gaurav; Cai, James; Wei, Xin; Lin, Tai-An; Heimbrook, David C; Packman, Kathryn; Boylan, John F

    2014-01-01

    As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment. PMID:24002693

  9. PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy.

    PubMed

    Malek, Anastasia; Czubayko, Frank; Aigner, Achim

    2008-02-01

    Gene targeting by RNA interference (RNAi) is mediated through small interfering RNA (siRNA), which, as plasmid DNA molecules, can be delivered into cells by polyethylenimines (PEI). Grafting with poly(ethylene glycol) has been introduced previously to improve PEI biocompatibility; however, data on the effects of PEGylation have been somewhat contradictory and various PEI(-PEG) need to be evaluated independently for DNA transfection and siRNA gene targeting efficacies. We directly compare plasmid DNA transfection and siRNA-mediated gene targeting efficacies, employing a larger set of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG; PEI(-PEG)) with different molecular weights and degrees of PEG substitution. We performed tissue culture-based bioassays on DNA transfection and siRNA-mediated targeting efficacies as well as on toxicity and cellular nucleic acid uptake, and, using sensitive assays based on radioactive labelling, physicochemically characterize the complexes regarding the degree of nucleic acid complexation and complex stabilities under various conditions. In contrast to the DNA transfection efficacy, siRNA-mediated gene targeting is much less dependent on the PEGylation of PEI or on the N/P ( = PEI nitrogen/nucleic acid phosphate) ratio. A more detailed analysis reveals that, in order to define optimal N/P ratios for DNA transfection, complex toxicities and nucleic acid uptake are the most critical parameters. In contrast, at optimal N/P ratios, complex stabilities and complexation efficacies determine PEI(-PEG)/DNA transfection efficacies and the major differences between various PEI(-PEG) are observed. All these parameters are less critical for PEI(-PEG)/siRNA gene targeting efficacy. Thus, our data lead to the distinction between three PEI(-PEG) groups, which relies on the differences in transfection rather than gene targeting efficacies, and which is correlated with the molecular weights and degrees of PEG substitution. In contrast to PEI

  10. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    PubMed Central

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

  11. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

    PubMed

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.

  12. Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution.

    PubMed

    Abdel-Wahab, Zeinab; Kalady, Matthew F; Emani, Sirisha; Onaitis, Mark W; Abdel-Wahab, Omar I; Cisco, Robin; Wheless, Lee; Cheng, Tsung-Yen; Tyler, Douglas S; Pruitt, Scott K

    2003-08-01

    Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.

  13. Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4.

    PubMed

    Cisco, Robin M; Abdel-Wahab, Zeinab; Dannull, Jens; Nair, Smita; Tyler, Douglas S; Gilboa, Eli; Vieweg, Johannes; Daaka, Yehia; Pruitt, Scott K

    2004-06-01

    Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

  14. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate.

    PubMed

    Wu, Kaimin; Xu, Jie; Liu, Mengyuan; Song, Wen; Yan, Jun; Gao, Shan; Zhao, Lingzhou; Zhang, Yumei

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.

  15. Virus-Like Particles of mRNA with Artificial Minimal Coat Proteins: Particle Formation, Stability, and Transfection Efficiency.

    PubMed

    Jekhmane, Shehrazade; de Haas, Rob; Paulino da Silva Filho, Omar; van Asbeck, Alexander H; Favretto, Marco Emanuele; Hernandez Garcia, Armando; Brock, Roland; de Vries, Renko

    2017-06-01

    RNA has enormous potential as a therapeutic, yet, the successful application depends on efficient delivery strategies. In this study, we demonstrate that a designed artificial viral coat protein, which self-assembles with DNA to form rod-shaped virus-like particles (VLPs), also encapsulates and protects mRNA encoding enhanced green fluorescent protein (EGFP) and luciferase, and yields cellular expression of these proteins. The artificial viral coat protein consists of an oligolysine (K12) for binding to the oligonucleotide, a silk protein-like midblock S10 = (GAGAGAGQ)10 that self-assembles into stiff rods, and a long hydrophilic random coil block C that shields the nucleic acid cargo from its environment. With mRNA, the C-S10-K12 protein coassembles to form rod-shaped VLPs each encapsulating about one to five mRNA molecules. Inside the rod-shaped VLPs, the mRNAs are protected against degradation by RNAses, and VLPs also maintain their shape following incubation with serum. Despite the lack of cationic surface charge, the mRNA VLPs transfect cells with both EGFP and luciferase, although with a much lower efficiency than obtained by a lipoplex transfection reagent. The VLPs have a negligible toxicity and minimal hemolytic activity. Our results demonstrate that VLPs yield efficient packaging and shielding of mRNA and create the basis for implementation of additional virus-like functionalities to improve transfection and cell specificity, such as targeting functionalities.

  16. Dendritic Cells Transfected with a DNA Construct Encoding Tumour-associated Antigen Epitopes Induce a Cytotoxic Immune Response Against Autologous Tumour Cells in a Culture of Mononuclear Cells from Colorectal Cancer Patients.

    PubMed

    Kulikova, E V; Kurilin, V V; Shevchenko, J A; Obleukhova, I A; Khrapov, E A; Boyarskikh, U A; Filipenko, M L; Shorokhov, R V; Yakushenko, V K; Sokolov, A V; Sennikov, S V

    2015-08-01

    Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour-associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA-transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic-assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA-transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin-positive lymphocytes. In addition, DNA-transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate-loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC-based vaccines.

  17. PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma

    PubMed Central

    Cai, Chenlei; Xie, Yuexia; Wu, liangliang; Chen, Xiaojing; Liu, Hongmei; Zhou, Yan; Zou, Hanbing; Liu, Dejun; Zhao, Yanan; Kong, Xianming; Liu, Peifeng

    2017-01-01

    Hepatic carcinoma (HCC) is a lethal disease associated with high morbidity and poor prognosis. Recently years, gene therapies have offered novel modalities to improve the prognosis of HCC patients. MicroRNA-99a (miR-99a) is frequently down-regulated in HCC, where it acts as a tumor suppressor. Therefore, we constructed monomethoxy (polyethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(L-lysine)-lactobionic acid- anti-vascular endothelial growth factor antibody (mPEG-PLGA-PLL-LA/VEGFab or PEAL-LA/VEGFab) nanoparticles (NPs) with highly specific targeting properties as carriers to restore the expression of miR-99a both in vitro and in vivo, to inhibit HCC progression. In vitro, PEAL-LA/VEGFab NPs showed more efficient delivery of miR-99a to HepG2 cells than the conventional transfection reagent LipofectamineTM2000 (Lip2000). The higher delivery efficiency associated with PEAL-LA/VEGFab NPs consequently resulted in down-regulation of target genes and suppression of the proliferation, migration and invasion of HepG2 cells. In vivo, miR-99a-PEAL-LA/VEGFab NPs inhibited tumor xenograft growth in HCC-bearing mice without causing obvious systemic toxicity. Our results demonstrate that PEAL-LA/VEGFab NPs selectively and effectively deliver miR-99a to HCC cells based on the double-targeting character of these nanoparticles, thereby offering potential for translation into effective clinical therapies for HCC. PMID:28387375

  18. Time-lapse imaging of mitosis after siRNA transfection.

    PubMed

    Mackay, Douglas R; Ullman, Katharine S; Rodesch, Christopher K

    2010-06-06

    Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

  19. Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

    SciTech Connect

    Zhou Hongsheng; Zhang Donghua . E-mail: hanson2008@gmail.com; Wang Yaya; Dai Ming; Zhang Lu; Liu Wenli; Liu Dan; Tan Huo; Huang Zhenqian

    2006-08-18

    CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

  20. Simultaneous activation of viral antigen-specific memory CD4+ and CD8+ T-cells using mRNA-electroporated CD40-activated autologous B-cells.

    PubMed

    Van den Bosch, Glenn A; Van Gulck, Ellen; Ponsaerts, Peter; Nijs, Griet; Lenjou, Marc; Apers, Ludwig; Kint, Ilse; Heyndrickx, Leo; Vanham, Guido; Van Bockstaele, Dirk R; Berneman, Zwi N; Van Tendeloo, Viggo F I

    2006-01-01

    Recently, it has become obvious that not only CD8 T-cells, but also CD4 T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 and CD8 T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-gamma-producing CD4 and CD8 autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 and CD8 T-cells. More detailed analysis of the activated interferon-gamma-producing CMV pp65 tetramer positive CD8 T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 and CD8 (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.

  1. Transfection of RNA from organ samples of infected animals represents a highly sensitive method for virus detection and recovery of classical swine fever virus.

    PubMed

    Meyer, Denise; Schmeiser, Stefanie; Postel, Alexander; Becher, Paul

    2015-01-01

    Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.

  2. Off Target, but Sequence-Specific, shRNA-Associated Trans-Activation of Promoter Reporters in Transient Transfection Assays

    PubMed Central

    Wan, Jun; Yerrabelli, Anitha; Berlinicke, Cindy; Kallman, Alyssa; Qian, Jiang; Zack, Donald J.

    2016-01-01

    Transient transfection promoter reporter assays are commonly used in the study of transcriptional regulation, and can be used to define and characterize both cis-acting regulatory sequences and trans-acting factors. In the process of using a variety of reporter assays designed to study regulation of the rhodopsin (rho) promoter, we discovered that rhodopsin promoter-driven reporter expression could be activated by certain species of shRNA in a gene-target-independent but shRNA sequence-specific manner, suggesting involvement of a specific shRNA associated pathway. Interestingly, the shRNA-mediated increase of rhodopsin promoter activity was synergistically enhanced by the rhodopsin transcriptional regulators CRX and NRL. Additionally, the effect was cell line-dependent, suggesting that this pathway requires the expression of cell-type specific factors. Since microRNA (miRNA) and interferon response-mediated processes have been implicated in RNAi off-target phenomena, we performed miRNA and gene expression profiling on cells transfected with shRNAs that do target a specific gene but have varied effects on rho reporter expression in order to identify transcripts whose expression levels are associated with shRNA induced rhodopsin promoter reporter activity. We identified a total of 50 miRNA species, and by microarray analysis, 320 protein-coding genes, some of which were predicted targets of the identified differentially expressed miRNAs, whose expression was altered in the presence of shRNAs that stimulated rhodopsin-promoter activity in a non-gene-targeting manner. Consistent with earlier studies on shRNA off-target effects, a number of interferon response genes were among those identified to be upregulated. Taken together, our results confirm the importance of considering off-target effects when interpreting data from RNAi experiments and extend prior results by focusing on the importance of including multiple and carefully designed controls in the design and

  3. A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated mRNA Transfection

    PubMed Central

    Williams, Damian J.; Puhl, Henry L.; Ikeda, Stephen R.

    2010-01-01

    Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K+ channel (GIRK4) and a dominant-negative G protein α-subunit mutant (GoA G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research. PMID:21267423

  4. Promoted adipogenesis of rat mesenchymal stem cells by transfection of small interfering RNA complexed with a cationized dextran.

    PubMed

    Nagane, Kentaro; Jo, Jun-ichiro; Tabata, Yasuhiko

    2010-01-01

    The objective of this study is to investigate the possibility of small interfering RNA (siRNA) complexed with a cationized dextran of nonviral carrier to biologically modify the adipogenesis extent of bone marrow-derived mesenchymal stem cells (MSC). Spermine was chemically introduced to the hydroxyl groups of dextran to prepare the cationized dextran (spermine-dextran). The spermine-dextran could form a complex with siRNA, and the physicochemical properties were changed by the molecular weight of dextran, the molar percentage of spermine introduced to dextran, and the molar ratio of nitrogen molecule of spermine-dextran to the phosphorous ones of siRNA (N/P ratio). The gene expression level of luciferase or green fluorescence protein was significantly suppressed by transfection with the complex of spermine-dextran and siRNA. The gene expression level by the complex decreased with an increase in the extent of complex internalized. Biochemical experiments revealed that culture in an adipogenic differentiation medium allowed MSC to differentiate into adipogenic cells. However, upon culturing with siRNA of anti-transcription coactivator containing PDZ-binding motif (TAZ) for osteogenic differentiation complexed with the spermine-dextran, the adipogenesis of MSC was further promoted. It is concluded that the spemine-dextran was a promising nonviral carrier to suppress the expression level of differentiation gene, resulting in the modification of cell differentiation direction.

  5. RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.

    PubMed

    Rakic, Rodolphe; Bourdon, Bastien; Hervieu, Magalie; Branly, Thomas; Legendre, Florence; Saulnier, Nathalie; Audigié, Fabrice; Maddens, Stéphane; Demoor, Magali; Galera, Philippe

    2017-08-24

    As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

  6. Effect of GPE-AGT nanoparticle shRNA transfection system mediated RNAi on early atherosclerotic lesion.

    PubMed

    Lu, Ping; Yuan, Lifen; Wang, Yuqiang; Du, Qin; Sheng, Jing

    2012-01-01

    To investigate the effects of RNA interference targeting AGT on early atherosclerotic lesion in the hypertensive state. Hypertension and atherosclerosis rats were treated with GPE nanoparticles carrying AGT shRNA. Systolic blood pressure and heart rate were measured for 2 consecutive weeks. Three days after treatment, the mRNA and protein expressions of AGT in the liver were measured by PCR and western blot assay, respectively. The blood levels of AGT and Ang II were determined by ELISA. H&E staining and electron microscopy were performed. Three days after AGT shRNA treatment, the mRNA and protein expressions of AGT in the liver were markedly reduced and the blood levels of AGT and Ang II dramatically decreased as compared to the remaining 3 groups (P < 0.05). Three days after AGT shRNA treatment, the blood pressure was reduced by 27 ± 4 mmHg when compared with that at baseline (P < 0.05). About 11 days after AGT shRNA treatment, the blood pressure began to increase. The blood pressure remained unchanged in the remaining 3 groups. Microscopy showed the atherosclerotic lesions were markedly attenuated in AGT shRNA treated rats but the liver and kidney functions remained stable (P > 0.05) when compared with the remaining 3 groups. Transfection with GPE nanoparticle carrying AGT shRNA can stably lower the blood pressure and improve the atherosclerotic lesions which lead to the delayed development of early atherosclerotic lesions in hypertension rats with concomitant atherosclerosis.

  7. Effect of GPE-AGT nanoparticle shRNA transfection system mediated RNAi on early atherosclerotic lesion

    PubMed Central

    Lu, Ping; Yuan, Lifen; Wang, Yuqiang; Du, Qin; Sheng, Jing

    2012-01-01

    Objective To investigate the effects of RNA interference targeting AGT on early atherosclerotic lesion in the hypertensive state. Methods Hypertension and atherosclerosis rats were treated with GPE nanoparticles carrying AGT shRNA. Systolic blood pressure and heart rate were measured for 2 consecutive weeks. Three days after treatment, the mRNA and protein expressions of AGT in the liver were measured by PCR and western blot assay, respectively. The blood levels of AGT and Ang II were determined by ELISA. H&E staining and electron microscopy were performed. Results Three days after AGT shRNA treatment, the mRNA and protein expressions of AGT in the liver were markedly reduced and the blood levels of AGT and Ang II dramatically decreased as compared to the remaining 3 groups (P < 0.05). Three days after AGT shRNA treatment, the blood pressure was reduced by 27 ± 4mmHg when compared with that at baseline (P < 0.05). About 11 days after AGT shRNA treatment, the blood pressure began to increase. The blood pressure remained unchanged in the remaining 3 groups. Microscopy showed the atherosclerotic lesions were markedly attenuated in AGT shRNA treated rats but the liver and kidney functions remained stable (P > 0.05) when compared with the remaining 3 groups. Conclusion Transfection with GPE nanoparticle carrying AGT shRNA can stably lower the blood pressure and improve the atherosclerotic lesions which lead to the delayed development of early atherosclerotic lesions in hypertension rats with concomitant atherosclerosis. PMID:22977667

  8. RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.

    PubMed

    Gruber, Jens; Boese, Guido; Tuschl, Thomas; Osborn, Mary; Weber, Klaus

    2004-07-01

    The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.

  9. Transfection of exogenous rotavirus rearranged RNA segments in cells infected with a WT rotavirus results in subsequent gene rearrangements.

    PubMed

    Duponchel, Sarah; Troupin, Cécile; Vu, Lan Trang; Schnuriger, Aurélie; Trugnan, Germain; Garbarg-Chenon, Antoine

    2014-09-01

    Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach. © 2014 The Authors.

  10. Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells.

    PubMed

    Hati Boruah, Jyoti Lakshmi; Ranjan, Rakesh; Gogoi, Hamen; Pandey, Saurabh Kumar; Kumar, Dharmendra; Phukan, Amlan Jyoti; Bori, Joygeswar; Sarkhel, Bikash Chandra

    2016-01-01

    Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique.

  11. cmRNA/lipoplex encapsulation in PLGA microspheres enables transfection via calcium phosphate cement (CPC)/PLGA composites.

    PubMed

    Utzinger, Maximilian; Jarzebinska, Anita; Haag, Nicolas; Schweizer, Martin; Winter, Gerhard; Dohmen, Christian; Rudolph, Carsten; Plank, Christian

    2017-03-10

    In this study lipoplexes containing chemically modified messenger RNA (cmRNA) were incorporated into poly (lactic-co-glycolic acid) (PLGA) microspheres via water-in-oil-in-water (W/O/W) double emulsion solvent evaporation technique. The nanoparticle encapsulation by microparticle formation was optimized to achieve lipoplex release and maximum transfection efficiency in surrounding cells. It was possible to adjust characteristic features in surface topology and size of the PLGA-microspheres by varying the extent of lipoplex loading into the polymer matrix. The partial release of lipids and mRNA out of the microparticle system, their accumulation in cells and the production of encoded protein were visualized via fluorescence microscopy. These bioactive microspheres, containing cmRNA bearing lipoplexes, were developed for the incorporation of a therapeutic component into injectable calcium phosphate cements (CPC). Due to the incorporation of PLGA/lipoplex microspheres as a degradable entity, the porosity of the cement phase could additionally be adjusted. This approach of complex nanoparticle incorporation into polymer/cement composites represents a promising example for combining transcript therapy with biomechanical engineering.

  12. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  13. A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

    PubMed Central

    Tagalakis, Aristides D; Castellaro, Sara; Zhou, Haiyan; Bienemann, Alison; Munye, Mustafa M; McCarthy, David; White, Edward A; Hart, Stephen L

    2015-01-01

    Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo. PMID:25878500

  14. Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells.

    PubMed

    Nowakowski, Adam; Andrzejewska, Anna; Boltze, Johannes; Nitzsche, Franziska; Cui, Li-Li; Jolkkonen, Jukka; Walczak, Piotr; Lukomska, Barbara; Janowski, Miroslaw

    2017-04-24

    Mesenchymal stem cells (MSCs) represent promising resource of cells for regenerative medicine in neurological disorders. However, efficient and minimally invasive methods of MSCs delivery to the brain still have to be developed. Intra-arterial route is very promising, but MSCs are missing machinery for diapedesis through blood-brain barrier. Thus, here we have tested a mRNA-based method to induce transient expression of ITGA4, an adhesion molecule actively involved in cell extravasation. We observed that transfection with an ITGA4-mRNA construct bearing a conventional cap analogue (7-methylguanosine) failed to produce ITGA4 protein, but exogenous ITGA4-mRNA was detected in transfected MSCs. This indicates that not transfection, but rather translation being the major roadblock. Stabilization of ITGA4-mRNA with SSB proteins resulted in ITGA4 protein synthesis in HEK293 cells only, whereas in MSCs, satisfactory results were obtained only after using an anti-reverse-cap-analogue (ARCA). The presence of ITGA4 protein in MSCs was transient and lasted for up to 24 h after transfection. Membranous location was confirmed by flow cytometry of viable non-permeabilized cells using anti-ITGA4 antibody. The mRNA-based expression of itga4 transgene is potentially sufficient for diapedesis after intra-arterial delivery. To conclude, mRNA-based engineering of stem cells is a rapid and integration-free method and attractive from the perspective of potential future clinical application.

  15. Transfection of infectious RNA and DNA/RNA layered vectors of semliki forest virus by the cell-penetrating peptide based reagent PepFect6.

    PubMed

    Pärn, Kalle; Viru, Liane; Lehto, Taavi; Oskolkov, Nikita; Langel, Ülo; Merits, Andres

    2013-01-01

    Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

  16. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    PubMed

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  17. Role of generation, architecture, pH and ionic strength on successful siRNA delivery and transfection by hybrid PPV-PAMAM dendrimers.

    PubMed

    Pavan, G M; Monteagudo, S; Guerra, J; Carrión, B; Ocaña, V; Rodríguez-Lopez, J; Danani, A; Pérez-Martínez, F C; Ceña, V

    2012-01-01

    Small interfering RNA (siRNA) constitutes an excellent way of knocking down genes. However, it requires the use of delivery systems to reach the target cells, especially to neuronal cells. Dendrimers are one of the most widely used synthetic nanocarriers for siRNA delivery. However, due to the complexity of the dendrimer-siRNA interactions, when a new dendritic carrier is designed it is difficult to predict its efficiency to bind and to deliver siRNA. At the same time it is not easy to understand the origin of eventual limited functionalities. We have modeled the interactions between two dendrimers (TDG-G1 and TDG-G2) and siRNA using molecular dynamics (MD) simulation. The results were compared to experimental physico-chemical parameters such as siRNA complexation, complex stability, size, and zeta potentials and biological effects such as down-regulation of a specific RNA expression in cortical neurons in culture. Data indicate that the combination of rigid core and flexible branches guarantees strong siRNA binding, which is important to have a good transfection profile. However, the successful nanocarrier for siRNA delivery (TDG-G1) is identified not only by a high affinity for siRNA, but by a favorable equilibrium between a strong binding and the ability to release siRNA to exert its biological action. The conditions under which the dendriplex is formed are also relevant for transfection efficiency and biological activity.

  18. Impact of the structure of biocompatible aliphatic polycarbonates on siRNA transfection ability.

    PubMed

    Frère, Antoine; Kawalec, Michal; Tempelaar, Sarah; Peixoto, Paul; Hendrick, Elodie; Peulen, Olivier; Evrard, Brigitte; Dubois, Philippe; Mespouille, Laetitia; Mottet, Denis; Piel, Géraldine

    2015-03-09

    RNAi therapeutics are promising therapeutic tools that have sparked the interest of many researchers. In an effort to provide a safe alternative to PEI, we have designed a series of new guanidinium- and morpholino-functionalized biocompatible and biodegradable polycarbonate vectors. The impact of different functions (morpholino-, guanidinium-, hydrophobic groups) of the architecture (linear homopolymer to dumbbell-shape) and of the molecular weight of these copolymers on their capacity to form polyplexes and to decrease the expression of two epigenetic regulators of gene expression, HDAC7 and HDAC5, was evaluated. The use of one of these polymers combining morpholine and guanidine functions at the ratio >1 and hydrophobic trimethylene carbonate groups showed a significant decrease of mRNA and protein level in HeLa cells, similar to PEI. These results highlight the potential of polycarbonate vectors for future in vivo application as an anticancer therapy.

  19. Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models.

    PubMed

    Mohan, Nishant; Chakrabarti, Mrinmay; Banik, Naren L; Ray, Swapan K

    2013-01-01

    Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

  20. Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

    PubMed

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation

    PubMed Central

    Bowles, Robert; Patil, Sonali; Pincas, Hanna; Sealfon, Stuart C.

    2014-01-01

    Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFN-β mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs. PMID:20875421

  2. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons

    PubMed Central

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I.; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the “in vivo” transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs. PMID:27047347

  3. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons.

    PubMed

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the "in vivo" transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs.

  4. Polymeric micelles self-assembled from amphiphilic polymers with twin disulfides used as siRNA carriers to enhance the transfection.

    PubMed

    Zhang, Can Yang; Peng, Shiyuan; Zhao, Bin; Luo, Wenji; Zhang, Lijuan

    2017-09-01

    The sequence-defined polycationic polymers with or without Cys-Arg-Cys motifs conjugated with targeting and shielding segments were synthesized as siRNA carriers via native chemical ligation (NCL) reaction. After purification, the structures and physicochemical characteristics were determined by a variety of experimental techniques. The particle size of siRNA/CRC-polymer polyplex was much smaller than that of polyplex without CRC motifs. The buffer capacity and siRNA binding ability of CRC motifs modified polymers were significantly improved, resulting from the twin disulfides and hexatomic ring formulation. The critical micelle concentrations of the polymers were <10mg/L, indicating formation of polymeric micelles and sufficient stability of the system. The CRC motifs modified polymers with folate targeted ligands exhibited a strongly enhanced cellular uptake than the negative control and the unmodified analogues. The results of gene transfection showed that the folate-PEG-ligated polymer modified with CRC motifs had much better gene transfection compared to the alanine-ended control and other analogues. Furthermore, they showed barely cytotoxicity. By the way, there was no distinctly improvement for pDNA transfection. All above results suggested that folate-PEG-ligated polymers modified with CRC motifs and their self-assembly polymeric micelles could be promising non-viral siRNA carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Identification of Cyclobutane Pyrimidine Dimer-Responsive Genes Using UVB-Irradiated Human Keratinocytes Transfected with In Vitro-Synthesized Photolyase mRNA.

    PubMed

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; van der Horst, Gijsbertus T J; Szegedi, Andrea; Horkay, Irén; Emri, Gabriella; Karikó, Katalin; Remenyik, Éva

    2015-01-01

    Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases.

  6. Transmission of HCV to a chimpanzee using virus particles produced in an RNA-transfected HepG2 cell culture.

    PubMed

    Dash, S; Kalkeri, G; McClure, H M; Garry, R F; Clejan, S; Thung, S N; Murthy, K K

    2001-10-01

    It was demonstrated previously that HepG2 cells produce negative strand RNA and virus-like particles after transfection with RNA transcribed from a full-length hepatitis C virus (HCV) cDNA clone [Dash et al. (1997) American Journal of Pathology, 151:363-373]. To determine in vivo infectivity of these in vitro synthesized viral particles, a chimpanzee was inoculated intravenously with HCV derived from HepG2 cells. The infected chimpanzee was examined serially for elevation of liver enzymes, for the presence of HCV RNA in the serum by reverse transcription nested polymerase chain reaction (RT-PCR), anti-HCV antibodies in the serum, and inflammation in the liver. The chimpanzee developed elevated levels of liver enzymes after the second week, but the levels fluctuated over a 10-week period. HCV RNA was detected in the serum of the chimpanzee at the second, seventh and ninth weeks after inoculation, and remained positive up to 25 weeks. Liver biopsies at Weeks 18 and 19 revealed of mild inflammation. Nucleotide sequence analysis of HCV recovered from the infected chimpanzee at the second and ninth weeks showed 100% sequence homology with the clone used for transfection studies. Serum anti-HCV antibodies were not detected by EIA during the 25 weeks follow-up period. These results suggest that intravenous administration of the virus-like particles derived from RNA-transfected HepG2 cells are infectious, and therefore, the pMO9.6-T7 clone is an infectious clone. These results provide new information that in vitro synthesized HCV particles produced from full-length HCV clone can cause infection in a chimpanzee. This study will facilitate the use of innovative approaches to the study of assembly of HCV particles and mechanisms of virus infectivity in cell culture.

  7. Adeno-associated virus-2-mediated TGF-β1 microRNA transfection inhibits adhesion formation after digital flexor tendon injury.

    PubMed

    Wu, Y F; Mao, W F; Zhou, Y L; Wang, X T; Liu, P Y; Tang, J B

    2016-02-01

    Adhesion formation after digital flexor tendon injury greatly affects gliding function of the tendon, which is a major clinical complication after hand surgery. Transforming growth factor beta 1 (TGF-β1) has a critical role in adhesion formation during tendon healing. Persistent regulation of TGF-β1 through application of microRNA (miRNA) specifically inhibiting the function of TGF-β1 (TGF-β1-miRNA) holds promise for treatment of such a complication. Adeno-associated virus (AAV) was used to transfer TGF-β1-miRNA to the chicken digital flexor tendons, which had been injured and surgically repaired. Four doses of AAV2-TGF-β1-miRNA (2 × 10¹¹, 2 × 10¹⁰, 2 × 10⁹ and 2 × 10⁸ vector genomes (vg)) were used to determine the transfection efficiency. At postoperative 3 weeks, we found a positive correlation between the administered AAV2-TGF-β1-miRNA doses and transfection efficiency. The transfection rate ranged from 10% to 77% as the doses increased. Production of TGF-β1 protein in the tendons decreased on increasing vector dosage. When 2 × 10¹¹ and 2 × 10¹⁰) vg were injected into the tendon, gliding excursion of the repaired tendon and work of flexion of chicken toes were significantly increased and adhesion score decreased 6 and 8 weeks later, indicating the improvement of tendon gliding and decreases in adhesion formations. However, the ultimate strength of the tendons transfected at the dose of 2 × 10¹⁰ vg was 12-24% lower than that of the control tendons. The results of this study demonstrate that application of TGF-β1-miRNA had a mixed impact on tendon healing: adhesion around the tendon is reduced but strength of the tendon healing is adversely affected. Future studies should aim at maintaining the beneficial effects of reducing tendon adhesions, while eliminating the adverse effects of decreasing the healing strength.

  8. 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.

  9. Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

    PubMed Central

    Proff, Julia; Schaft, Niels; Dörrie, Jan; Full, Florian; Ensser, Armin; Muller, Yves A.; Cerwenka, Adelheid; Abken, Hinrich; Parolini, Ornella; Ambros, Peter F.; Kovar, Heinrich; Holter, Wolfgang

    2012-01-01

    We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection. PMID:22355347

  10. Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

    PubMed

    Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

    2017-01-01

    Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10(6) to approximately 10(8) cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

  11. In vivo transfection of manganese superoxide dismutase gene or nuclear factor κB shRNA in nodose ganglia improves aortic baroreceptor function in heart failure rats.

    PubMed

    Zhang, Dongze; Liu, Jinxu; Tu, Huiyin; Muelleman, Robert L; Cornish, Kurtis G; Li, Yu-Long

    2014-01-01

    Arterial baroreflex sensitivity is attenuated in chronic heart failure (CHF) state, which is associated with cardiac arrhythmias and sudden cardiac death in patients with CHF. Our previous study showed that CHF-induced sodium channel dysfunction in the baroreceptor neurons was involved in the blunted baroreflex sensitivity in CHF rats. Mitochondria-derived superoxide overproduction decreased expression and activation of the sodium channels in the baroreceptor neurons from CHF rats. However, the molecular mechanisms responsible for the sodium channel dysfunction in the baroreceptor neurons from CHF rats remain unknown. We tested the involvement of nuclear factor κB (NFκB) in the sodium channel dysfunction and evaluated the effects of in vivo transfection of manganese superoxide dismutase gene and NFκB shRNA on the baroreflex function in CHF rats. CHF was developed at 6 to 8 weeks after left coronary artery ligation in adult rats. Western blot and chromatin immunoprecipitation data showed that phosphorylated NFκB p65 and ability of NFκB p65 binding to the sodium channel promoter were increased in the nodose ganglia from CHF rats. In vivo transfection of adenoviral manganese superoxide dismutase gene or lentiviral NFκB p65 shRNA into the nodose ganglia partially reversed CHF-reduced sodium channel expression and cell excitability in the baroreceptor neurons and improved CHF-blunted arterial baroreflex sensitivity. Additionally, transfection of adenoviral manganese superoxide dismutase also inhibited the augmentation of phosphorylated NFκB p65 in the nodose neurons from CHF rats. The present study suggests that superoxide-NFκB signaling contributes to CHF-induced baroreceptor dysfunction and resultant impairment of baroreflex function.

  12. Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum.

    PubMed

    Materna, Stefan C; Marwan, Wolfgang

    2005-02-01

    We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.

  13. Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells.

    PubMed

    Kyte, Jon Amund; Aamdal, Steinar; Dueland, Svein; Sæbøe-Larsen, Stein; Inderberg, Else Marit; Madsbu, Ulf Erik; Skovlund, Eva; Gaudernack, Gustav; Kvalheim, Gunnar

    2016-01-01

    The most effective anticancer immune responses are probably directed against patient-specific neoantigens. We have developed a melanoma vaccine targeting this individual mutanome based on dendritic cells (DCs) loaded with autologous tumor-mRNA. Here, we report a phase I/II trial evaluating toxicity, immune response and clinical outcome in 31 metastatic melanoma patients. The first cohort (n = 22) received the vaccine without any adjuvant; the next cohort (n = 9) received adjuvant IL2. Each subject received four weekly intranodal or intradermal injections, followed by optional monthly vaccines. Immune response was evaluated by delayed-type hypersensitivity (DTH), T cell proliferation and cytokine assays. Data were collected for 10 y after inclusion of the last patient. No serious adverse events were detected. In the intention-to-treat-cohort, we demonstrated significantly superior survival compared to matched controls from a benchmark meta-analysis (1 y survival 43% vs. 24%, 2 y 23% vs. 6.6%). A tumor-specific immune response was demonstrated in 16/31 patients. The response rate was higher after intradermal than intranodal vaccination (80% vs. 38%). Immune responders had improved survival compared to non-responders (median 14 mo vs. 6 mo; p = 0.030), and all eight patients surviving >20 mo were immune responders. In addition to the tumor-specific response, most patients developed a response against autologous DC antigens. The cytokine profile was polyfunctional and did not follow a Th1/Th2 dichotomy. We conclude that the favorable safety profile and evidence of a possible survival benefit warrant further studies of the RNA/DC vaccine. The vaccine appears insufficient as monotherapy, but there is a strong rationale for combination with checkpoint modulators.

  14. Modification of tumor cell exosome content by transfection with wt-p53 and microRNA-125b expressing plasmid DNA and its effect on macrophage polarization

    PubMed Central

    Trivedi, M; Talekar, M; Shah, P; Ouyang, Q; Amiji, M

    2016-01-01

    Exosomes are responsible for intercellular communication between tumor cells and others in the tumor microenvironment. These microvesicles promote oncogensis and can support towards metastasis by promoting a pro-tumorogenic environment. Modifying the exosomal content and exosome delivery are emerging novel cancer therapies. However, the clinical translation is limited due to feasibility of isolating and delivery of treated exosomes as well as an associated immune response in patients. In this study, we provide proof-of-concept for a novel treatment approach for manipulating exosomal content by genetic transfection of tumor cells using dual-targeted hyaluronic acid-based nanoparticles. Following transfection with plasmid DNA encoding for wild-type p53 (wt-p53) and microRNA-125b (miR-125b), we evaluate the transgene expression in the SK-LU-1 cells and in the secreted exosomes. Furthermore, along with modulation of wt-p53 and miR-125b expression, we also show that the exosomes (i.e., wt-p53/exo, miR-125b/exo and combination/exo) have a reprogramed global miRNA profile. The miRNAs in the exosomes were mainly related to the activation of genes associated with apoptosis as well as p53 signaling. More importantly, these altered miRNA levels in the exosomes could mediate macrophage repolarization towards a more pro-inflammatory/antitumor M1 phenotype. However, further studies, especially in vivo studies, are warranted to assess the direct influence of such macrophage reprogramming on cancer cells and oncogenesis post-treatment. The current study provides a novel platform enabling the development of therapeutic strategies affecting not only the cancer cells but also the tumor microenvironment by utilizing the ‘bystander effect' through genetic transfer with secreted exosomes. Such modification could also support antitumor environment leading to decreased oncogenesis. PMID:27500388

  15. Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA.

    PubMed

    Gioio, Anthony E; Lavina, Zeno Scotto; Jurkovicova, Dana; Zhang, Hengshan; Eyman, Maria; Giuditta, Antonio; Kaplan, Barry B

    2004-08-01

    It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.

  16. Targeted delivery of biodegradable nanoparticles with ultrasound-targeted microbubble destruction-mediated hVEGF-siRNA transfection in human PC-3 cells in vitro.

    PubMed

    Li, Yun-Hua; Shi, Qiu-Sheng; Du, Jing; Jin, Li-Fang; Du, Lian-Fang; Liu, Pei-Feng; Duan, You-Rong

    2013-01-01

    A potentially viable approach for treating late-stage prostate cancer is gene therapy. Successful gene therapy requires safe and efficient delivery systems. In this study, we report the efficient delivery of small interfering RNA (siRNA) via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. On the basis of previous findings, cyclic Arg-Gly-Asp (cRGD) peptides were conjugated to NPs to recognize the target site, integrin αvβ3, expressed in high levels in PC-3 prostate cancer cells. The suppression of angiogenesis by the downregulation of vascular endothelial growth factor (VEGF) expression has been widely used to inhibit the growth of malignant tumors. In our study, human VEGF (hVEGF)-siRNA was encapsulated in NPs to inhibit VEGF expression in PC-3 cells. Concurrently, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for the delivery of siRNA-loaded NPs. Our results showed low cytotoxicity and high gene transfection efficiency, demonstrating that the targeted delivery of biodegradable NPs with UTMD may be potentially applied as new vector system for gene delivery.

  17. Co-transfection and tandem transfection of HEK293A cells for overexpression and RNAi experiments.

    PubMed

    Xie, Zhen-Li; Shao, Shu-Li; Lv, Jian-Wei; Wang, Chang-He; Yuan, Cheng-Zhi; Zhang, Wei-Wei; Xu, Xing-Jun

    2011-03-01

    pIRES2-EGFP was employed and a non-target shRNA expressing plasmid was constructed to simulate overexpression and RNAi (RNA interference) experiments. Transfection of pIRES2-EGFP into HEK293A cells by cationic lipids VigoFect demonstrated that transfection efficiency increased in a dose-dependent manner with amount of DNA plasmid used, and optimal transfection time and cell density should be identified to reach a compromise of higher transfection efficiency and lower toxicity. Co-transfection experiments indicated that the two co-transfected plasmids were equivalently delivered into the same cells, and the co-transfection efficiency was rarely affected by cell density and proportion of the two plasmids. However, plasmid-receipted cells seemed indisposed to accept plasmid again during the second transfection, and very low co-transfection efficiency was observed in tandem transfection.

  18. Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Choubey, Amit

    performing a 15 mus all-atom MD simulation of a DPPC-CHOL bilayer. We find that the CHOL flip-flop rates are on the sub microsecond timescale. These results are verified by performing various independent parallel replica (PR) simulations. Our PR simulations provide significant boost in sampling of the flip-flop events. We observe that the CHOL flip-flop can induce membrane order, regulate membrane-bending energy, and facilitate membrane relaxation. The rapid flip-flop rates reported here have important implications for the role of CHOL in mechanical properties of cell membranes, formation of domains, and maintaining CHOL concentration asymmetry in plasma membrane. Our PR approach can reach submillisecond time scales and bridge the gap between MD simulations and Nuclear Magnetic Resonance (NMR) experiments on CHOL flip-flop dynamics in membranes. The last project deals with transfection barriers encountered by a bare small interfering RNA (siRNA) in a phospholipid bilayer. SiRNA molecules play a pivotal role in therapeutic applications. A key limitation to the widespread implementation of siRNA-based therapeutics is the difficulty of delivering siRNA-based drugs to cells. We have examined structural and mechanical barriers to siRNA passage across a phospholipid bilayer using all-atom MD simulations. We find that the electrostatic interaction between the anionic siRNA and head groups of phospholipid molecules induces a phase transformation from the liquid crystalline to ripple phase. Steered MD simulations reveal that the siRNA transfection through the ripple phase requires a force of ˜ 1.5 nN.

  19. Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma

    PubMed Central

    Panjwani, M Kazim; Smith, Jenessa B; Schutsky, Keith; Gnanandarajah, Josephine; O'Connor, Colleen M; Powell, Daniel J; Mason, Nicola J

    2016-01-01

    Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans. PMID:27401141

  20. Improving the osteogenesis of human bone marrow mesenchymal stem cell sheets by microRNA-21-loaded chitosan/hyaluronic acid nanoparticles via reverse transfection

    PubMed Central

    Wang, Zhongshan; Wu, Guangsheng; Wei, Mengying; Liu, Qian; Zhou, Jian; Qin, Tian; Feng, Xiaoke; Liu, Huan; Feng, Zhihong; Zhao, Yimin

    2016-01-01

    Cell sheet engineering has emerged as a novel approach to effectively deliver seeding cells for tissue regeneration, and developing human bone marrow mesenchymal stem cell (hBMMSC) sheets with high osteogenic ability is a constant requirement from clinics for faster and higher-quality bone formation. In this work, we fabricated biocompatible and safe chitosan (CS)/hyaluronic acid (HA) nanoparticles (NPs) to deliver microRNA-21 (miR-21), which has been proved to accelerate osteogenesis in hBMMSCs; then, the CS/HA/miR-21 NPs were cross-linked onto the surfaces of culture plates with 0.2% gel solution to fabricate miR-21-functionalized culture plates for reverse transfection. hBMMSC sheets were induced continuously for 14 days using a vitamin C-rich method on the miR-21-functionalized culture plates. For the characterization of CS/HA/miR-21 NPs, the particle size, zeta potential, surface morphology, and gel retardation were sequentially investigated. Then, the biological effects of hBMMSC sheets on the miR-21-functionalized culture plates were evaluated. The assay results demonstrated that the hBMMSC sheets could be successfully induced via the novel reverse transfection approach, and miR-21 delivery significantly enhanced the in vitro osteogenic differentiation of hBMMSC sheets in terms of upregulating calcification-related gene expression and enhancing alkaline phosphatase production, collagen secretion, and mineralized nodule formation. The enhanced osteogenic activity of hBMMSC sheets might promisingly lead to more rapid and more robust bone regeneration for clinical use. PMID:27274237

  1. Three-year Follow up of GMCSF/bi-shRNA(furin) DNA-transfected Autologous Tumor Immunotherapy (Vigil) in Metastatic Advanced Ewing's Sarcoma.

    PubMed

    Ghisoli, Maurizio; Barve, Minal; Mennel, Robert; Lenarsky, Carl; Horvath, Staci; Wallraven, Gladice; Pappen, Beena O; Whiting, Sam; Rao, Donald; Senzer, Neil; Nemunaitis, John

    2016-08-01

    Ewing's sarcoma is a devastating rare pediatric cancer of the bone. Intense chemotherapy temporarily controls disease in most patients at presentation but has limited effect in patients with progressive or recurrent disease. We previously described preliminary results of a novel immunotherapy, FANG (Vigil) vaccine, in which 12 advanced stage Ewing's patients were safely treated and went on to achieve a predicted immune response (IFNγ ELISPOT). We describe follow-up through year 3 of a prospective, nonrandomized study comparing an expanded group of Vigil-treated advanced disease Ewing's sarcoma patients (n = 16) with a contemporaneous group of Ewing's sarcoma patients (n = 14) not treated with Vigil. Long-term follow-up results show a survival benefit without evidence of significant toxicity (no ≥ grade 3) to Vigil when administered once monthly by intradermal injection (1 × 10e(6) cells/injection to 1 × 10e(7) cells/injection). Specifically, we report a 1-year actual survival of 73% for Vigil-treated patients compared to 23% in those not treated with Vigil. In addition, there was a 17.2-month difference in overall survival (OS; Kaplan-Meier) between the Vigil (median OS 731 days) and no Vigil patient groups (median OS 207 days). In conclusion, these results supply the rational for further testing of Vigil in advanced stage Ewing's sarcoma.

  2. Atelocollagen-mediated in vivo siRNA transfection in ovarian carcinoma is influenced by tumor site, siRNA target and administration route.

    PubMed

    Meryet-Figuière, Matthieu; Lecerf, Charlotte; Varin, Emilie; Coll, Jean-Luc; Louis, Marie-Hélène; Dutoit, Soizic; Giffard, Florence; Blanc-Fournier, Cécile; Hedir, Siham; Vigneron, Nicolas; Brotin, Emilie; Pelletier, Laurent; Josserand, Véronique; Denoyelle, Christophe; Poulain, Laurent

    2017-10-01

    Ovarian cancer is the leading cause of death from gynecological malignancies worldwide, and innate or acquired chemoresistance of ovarian cancer cells is the major cause of therapeutic failure. It has been demonstrated that the concomitant inhibition of Bcl-xL and Mcl-1 anti-apoptotic activities is able to trigger apoptosis in chemoresistant ovarian cancer cells. In this context, siRNA-mediated Bcl‑xL and Mcl-1 inhibition constitutes an appealing strategy by which to eliminate chemoresistant cancer cells. However, the safest and most efficient way to vectorize siRNAs in vivo is still under debate. In the present study, using in vivo bioluminescence imaging, we evaluated the interest of atelocollagen to vectorize siRNAs by intraperitoneal (i.p.) or intravenous (i.v.) administration in 2 xenografted ovarian cancer models (peritoneal carcinomatosis and subcutaneous tumors in nude mice). Whereas i.p. administration of atelocollagen-vectorized siRNA in the peritoneal carcinomatosis model did not induce any gene downregulation, a 70% transient downregulation of luciferase expression was achieved after i.v. injection of atelocollagen-vectorized siRNA in the subcutaneous (s.c.) model. However, the use of siRNA targeting Bcl-xL or Mcl-1 did not induce target-specific downregulation in vivo in nude mice. Our results therefore show that atelocollagen complex formulation, the administration route, tumor site and the identity of the siRNA target influence the efficiency of atelocollagen‑mediated siRNA delivery.

  3. Long-term marker-free multiphoton imaging, targeted transfection, optical cleaning of stem cell clusters, and optical transport of microRNA with extreme ultrashort laser pulses

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada; Földes-Papp, Zeno; Kostner, Gerhard M.; König, Karsten

    2010-02-01

    The novel utrashort femtosecond laser scanning microscope FemtOgene (JenLab GmbH, Germany) with 12 femtoseconds at the focal plane have been employed in marker-free imaging and optical manipulation of stem cells as well as for the non-contact introduction of microRNA in cancer cells. Human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells have been investigated by femtosecond laser multiphoton microscopy. Autofluorescence based on NAD(P)H and flavoproteins and second harmonic generation due to collagen have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. Major emission peaks at 460 nm and 530 nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured in a variety of stem cells using spectral imaging and time-correlated single photon counting. During differentiation, the ratios of bound to free NAD(P)H and NAD(P)H/flavoproteins changed. In addition, the biosynthesis of lipids and collagen was detected over a long period of time of up to 5 weeks. Nanoprocessing was performed with 12 femtosecond laser pulses and low picojoule pulse energies to realize targeted transfection and optical cleaning of human adult stem cell populations. Multiphoton sub-20fs microscopes may become novel non-invasive tools for marker-free optical stem cell characterization, for on-line monitoring of differentiation within a three-dimensional microenvironment, and for optical manipulation.

  4. NITRIC OXIDE PRODUCTION AND iNOS mRNA EXPRESSION IN IFN-8-STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH iNOS siRNAs

    USDA-ARS?s Scientific Manuscript database

    Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-' induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biological pathway i...

  5. Optimization of renal transfection using a renal suction-mediated transfection method in mice.

    PubMed

    Taniguchi, Yota; Kawakami, Shigeru; Fuchigami, Yuki; Oyama, Natsuko; Yamashita, Fumiyoshi; Konishi, Satoshi; Shimizu, Kazunori; Hashida, Mitsuru

    2016-01-01

    We previously developed a suction-mediated transfection method in mice. The purpose of this study was to optimize the suction-mediated transfection conditions using a pressure-controlled computer system for efficient and safe kidney-targeted gene delivery in mice. Naked pCMV-Luc was injected into the tail vein in mice, and then the right kidney was suctioned by a device of the suction pressure-controlled system. The effects of renal transfection conditions, such as the suction pressure degree, suction pressure waveform and device area were evaluated by measuring luciferase expression. In addition, renal injury was examined. The renal suction-mediated transfection method at -30 kPa showed high transgene expression. The renal suction waveform did not affect the transfection activity. Under the optimized conditions, the high transgene expression was mostly observed at the renal suctioned site. The transfection conditions used did not induce histological defects or increases in two renal injury biomarkers (Kidney injury molecule-1 mRNA and Clusterin mRNA). We have clarified the transfection conditions for efficient and safe transfection in the kidney using the suction-mediated transfection method in mice.

  6. RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma.

    PubMed

    Harrer, Dennis C; Simon, Bianca; Fujii, Shin-Ichiro; Shimizu, Kanako; Uslu, Ugur; Schuler, Gerold; Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

    2017-08-17

    Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA. PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8(+) T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed. Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8(+) MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8(+) T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells. We

  7. Enhanced delivery of PEAL nanoparticles with ultrasound targeted microbubble destruction mediated siRNA transfection in human MCF-7/S and MCF-7/ADR cells in vitro

    PubMed Central

    Teng, Yanwei; Bai, Min; Sun, Ying; Wang, Qi; Li, Fan; Xing, Jinfang; Du, Lianfang; Gong, Tao; Duan, Yourong

    2015-01-01

    The gene knockdown activity of small interfering RNA (siRNA) has led to their use as potential therapeutics for a variety of diseases. However, successful gene therapy requires safe and efficient delivery systems. In this study, we choose mPEG-PLGA-PLL nanoparticles (PEAL NPs) with ultrasound targeted microbubble destruction (UTMD) to efficiently deliver siRNA into cells. An emulsification-solvent evaporation method was used to prepare siRNA-loaded PEAL NPs. The NPs possessed an average size of 132.6±10.3 nm (n=5), with a uniform spherical shape, and had an encapsulation efficiency (EE) of more than 98%. As demonstrated by MTT assay, neither PEAL NPs nor siRNA-loaded PEAL NPs showed cytotoxicity even at high concentrations. The results of cellular uptake showed, with the assistance of UTMD, the siRNA-loaded PEAL NPs can be effectively internalized and can subsequently release siRNA in cells. Taken together, PEAL NPs with UTMD may be highly promising for siRNA delivery, making it possible to fully exploit the potential of siRNA-based therapeutics. PMID:26346350

  8. 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.

  9. Phase II Study of Autologous Monocyte-Derived mRNA Electroporated Dendritic Cells (TriMixDC-MEL) Plus Ipilimumab in Patients With Pretreated Advanced Melanoma.

    PubMed

    Wilgenhof, Sofie; Corthals, Jurgen; Heirman, Carlo; van Baren, Nicolas; Lucas, Sophie; Kvistborg, Pia; Thielemans, Kris; Neyns, Bart

    2016-04-20

    Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic mRNA (TriMixDC-MEL) are immunogenic and have antitumor activity as a monotherapy in patients with pretreated advanced melanoma. Ipilimumab, an immunoglobulin G1 monoclonal antibody directed against the cytotoxic T-lymphocyte-associated protein 4 receptor that counteracts physiologic suppression of T-cell function, improves the overall survival of patients with advanced melanoma. This phase II study investigated the combination of TriMixDC-MEL and ipilimumab in patients with pretreated advanced melanoma. Thirty-nine patients were treated with TriMixDC-MEL (4 × 10(6) cells administered intradermally and 20 × 10(6) cells administered intravenously) plus ipilimumab (10 mg/kg every 3 weeks for a total of four administrations, followed by maintenance therapy every 12 weeks in patients who remained progression free). Six-month disease control rate according to the immune-related response criteria served as the primary end point. The 6-month disease control rate was 51% (95% CI, 36% to 67%), and the overall tumor response rate was 38% (including eight complete and seven partial responses). Seven complete responses and one partial tumor response are ongoing after a median follow-up time of 36 months (range, 22 to 43 months). The most common treatment-related adverse events (all grades) consisted of local DC injection site skin reactions (100%), transient post-DC infusion chills (38%) and flu-like symptoms (84%), dermatitis (64%), hepatitis (13%), hypophysitis (15%), and diarrhea/colitis (15%). Grade 3 or 4 immune-related adverse events occurred in 36% of patients. There was no grade 5 adverse event. The combination of TriMixDC-MEL and ipilimumab is tolerable and results in an encouraging rate of highly durable tumor responses in patients with pretreated advanced melanoma. © 2016 by American Society of Clinical Oncology.

  10. Fibre based cellular transfection.

    PubMed

    Tsampoula, X; Taguchi, K; Cizmár, T; Garces-Chavez, V; Ma, N; Mohanty, S; Mohanty, K; Gunn-Moore, F; Dholakia, K

    2008-10-13

    Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

  11. Disassembly of micelle-like polyethylenimine nanocomplexes for siRNA delivery: High transfection efficiency and reduced toxicity achieved by simple reducible lipid modification.

    PubMed

    Guo, Gaoyang; Tortorella, Micky; Zhang, Biliang; Wang, Yunbing

    2017-10-15

    Amphiphilic compounds consisting of polycations and lipid segments are well established as building blocks for the construction of siRNA carriers. They are capable of forming nanoparticles with high-affinity positive charges for siRNA in aqueous media due to their intra- and/or intermolecular hydrophobic and electrostatic interactions. Unfortunately, safety and efficiency of lipid-modified polycations as the two great challenges to the clinical application need to be improved. Beyond that, the role of the hydrophobic segment in the process of siRNA delivery is elusive. Herein, in this study, branched polyethylenimine with a molecular weight of 600 (bPEI600) was grafted with reducible lipids via Michael addition reaction between amines and alkyl acrylates. Reducible amphiphilic polyethylenimines (PEIs) were able to condense siRNA into nanoparticles and disassemble under the reductive environment. Investigations with these materials in vitro revealed that the polymers with higher grafting degree provided high luciferase knockdown efficacies even at lower N/P ratios and the polymers with longer lipid chain displayed greater cellular uptake rate. Interestingly, the polymers with lower grafting degree had efficient cellular uptake than native bPEI600, although their in luciferase knockdown assays were most likely inefficient. The inconsistency between the cellular uptake profile and silencing efficacy proved that the intracellular trafficking of siRNA was a bottleneck for siRNA delivery with some polymers prepared in this study. As expected, reducible lipid-modified PEIs were equally efficient and much less toxic compared to non-reducible counterparts and might provide broader therapeutic windows. These findings showed the feasibility of reducible lipid-modified PEIs as carriers for therapeutic siRNA. Copyright © 2017. Published by Elsevier Inc.

  12. Graphene based gene transfection

    NASA Astrophysics Data System (ADS)

    Feng, Liangzhu; Zhang, Shuai; Liu, Zhuang

    2011-03-01

    Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI

  13. 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.

  14. Enhancement of the efficiency of femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

    Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput microfluidic optical transfection devices.

  15. Enhancement of the efficiency of femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

    Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput microfluidic optical transfection devices.

  16. Expression and analysis of a split premature termination codon in FGG responsible for congenital afibrinogenemia: escape from RNA surveillance mechanisms in transfected cells.

    PubMed

    Neerman-Arbez, Marguerite; Germanos-Haddad, Myrna; Tzanidakis, Konstantinos; Vu, Dung; Deutsch, Samuel; David, Armelle; Morris, Michael A; de Moerloose, Philippe

    2004-12-01

    Congenital afibrinogenemia, the most severe form of fibrinogen deficiency, is characterized by the complete absence of fibrinogen. The disease is caused by mutations in 1 of the 3 fibrinogen genes FGG, FGA, and FGB, clustered on the long arm of human chromosome 4. The majority of cases are due to null mutations in the FGA gene although one would expect the 3 genes to be equally implicated. However, most patients studied so far are white, and therefore the identification of causative mutations in non-European families is necessary to establish if this finding holds true in all ethnic groups. In this study, we report the identification of a novel nonsense mutation (Arg134Xaa) in the FGG gene responsible for congenital afibrinogenemia in 10 patients from Lebanon. Expression studies in COS-7 cells demonstrated that the Arg134Xaa codon, which is encoded by adjacent exons (TG-intron 4-A) affected neither mRNA splicing nor stability, but led to the production of an unstable, severely truncated fibrinogen gamma chain that is not incorporated into a functional fibrinogen hexamer.

  17. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    PubMed

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

  18. 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.

  19. 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.

  20. 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.

  1. Transfection of Platyhelminthes.

    PubMed

    Moguel, Bárbara; Bobes, Raúl J; Carrero, Julio C; Laclette, Juan P

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  2. Transfection of Platyhelminthes

    PubMed Central

    Moguel, Bárbara; Bobes, Raúl J.; Carrero, Julio C.; Laclette, Juan P.

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species. PMID:26090388

  3. Lipid-based transfection reagents can interfere with cholesterol biosynthesis.

    PubMed

    Danielli, Mauro; Marinelli, Raúl A

    2016-02-15

    Lipid-based transfection reagents are widely used for delivery of small interfering RNA into cells. We examined whether the commonly used commercial transfection reagents DharmaFECT-4 and Lipofectamine 2000 can interfere with lipid metabolism by studying cholesterogenesis. Cholesterol de novo synthesis from [(14)C]acetate was assessed in human hepatocyte-derived Huh-7 cells. The results revealed that DharmaFECT, but not Lipofectamine, markedly inhibited cholesterol biosynthesis by approximately 70%. Cell viability was not significantly altered. These findings suggest that caution is required in the choice of certain lipid-based transfection reagents for gene silencing experiments, particularly when assessing cholesterol metabolism.

  4. Electroporation for Transfection and Differentiation of Dental Pulp Stem Cells

    PubMed Central

    Rabie, Bakr M.

    2013-01-01

    Abstract Target gene delivery is needed to induce cellular differentiation or a specific therapeutic effect. Electroporation is a relatively safe and simple technique to deliver nucleic acids to the cell that acts by rendering cells transiently permeable using short periods of high voltage. In stem cell research, human dental pulp stem cells (hDPSCS) are highly accessible, and they exhibit broad differentiation potential. Until now, no studies have attempted to optimize electroporation parameters for DPSCs with respect to transfection efficiency and viability. In this study, we aimed to optimize transfection of DPSCs through varying different electroporation parameters, including voltage, mode of pulsation, and the number of pulses. As positive control, we used commonly utilized the chemical transfection reagents Lipofectamine 2000 and FuGene 6. In addition, we used our newly optimized transfection conditions to transfect hDPSCs with a functional chondrogenic transgene. We obtained higher transfection efficiency and cell viability with these electroporation conditions compared to controls. The highest transfection efficiency (63.81±4.72%) was achieved with 100 V, 20 msec, one-pulse square-wave condition. Among chemical transfection groups, FuGene 6 showed the highest cell viability at all tested transfection ratios, while Lipofectamine 2000 showed the highest transfection efficiency (19.23±3.19%) using 1:1 DNA (μg):Lipofectamine (μL). Transfected DPSCs functionally expressed the transforming growth factor β-3 chondrogenic transgene on the mRNA level as detected by real-time polymerase chain reaction and on the protein level as detected by Western blot analysis. An increase in various chondrogenic markers was also found when studying mRNA expression in transfected cells. In conclusion, the results of our study demonstrate optimal electroporation and chemical transfection reagent conditions for hDPSCs, and, subsequently, we provide proof of concept for

  5. Autologous Therapies in Dermatology

    PubMed Central

    Kumar, Sumir; Mahajan, Bharat Bhushan; Singh, Amarbir

    2014-01-01

    Autologous therapy is a therapeutic intervention that uses an individual’s cells or tissues, which are processed outside the body, and reintroduced into the donor. This emerging field presently represents a mere tip of the iceberg with much knowledge and applications yet to be discovered. It, being free from risks of hypersensitivity reactions and transmission of infectious agents, has been explored in various fields, such as plastic surgery, orthopedics, and dermatology. This review article focuses on various forms of autologous therapies used in dermatology along with their applications and mechanisms of action. PMID:25584137

  6. A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

    PubMed

    Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

    2006-09-01

    Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

  7. 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

  8. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  9. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    PubMed

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  10. Autologous Costochondral Microtia Reconstruction.

    PubMed

    Patel, Sapna A; Bhrany, Amit D; Murakami, Craig S; Sie, Kathleen C Y

    2016-04-01

    Reconstruction with autologous costochondral cartilage is one of the mainstays of surgical management of congenital microtia. We review the literature, present our current technique for microtia reconstruction with autologous costochondral graft, and discuss the evolution of our technique over the past 20 years. We aim to minimize donor site morbidity and create the most durable and natural appearing ear possible using a stacked framework to augment the antihelical fold and antitragal-tragal complex. Assessment of outcomes is challenging due to the paucity of available objective measures with which to evaluate aesthetic outcomes. Various instruments are used to assess outcomes, but none is universally accepted as the standard. The challenges we continue to face are humbling, but ongoing work on tissue engineering, application of 3D models, and use of validated questionnaires can help us get closer to achieving a maximal aesthetic outcome. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  11. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  12. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  13. Angiogenesis in rat uterine cicatrix after injection of autologous bone marrow mesenchymal stem cells.

    PubMed

    Maiborodin, I V; Yakimova, N V; Matveyeva, V A; Pekarev, O G; Maiborodina, E I; Pekareva, E O

    2011-04-01

    Results of injection of autologous bone marrow mesenchymal stem cells with transfected GFP gene into the rat uterine horn cicatrix were studied by light microscopy. Large groups of blood vessels with blood cells inside were seen after injection of autologous bone marrow cells into the cicatrix on the right horn, formed 2 months after its ligation; no groups of vessels of this kind were found in the cicatrix in the contralateral horn. Examination of unstained sections in reflected UV light showed sufficiently bright fluorescence in the endothelium and outer vascular membrane in the uterine horn cicatrix only on the side of injection. Hence, autologous mesenchymal stem cells injected into the cicatrix formed the blood vessels due to differentiation into endotheliocytes and pericytes. The expression of GFP gene not only in the vascular endothelium, but also in vascular outer membranes indicated that autologous mesenchymal stem cells differentiated in the endothelial and pericytic directions.

  14. 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.

  15. 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

  16. What is autologous blood transfusion?

    PubMed

    Sansom, A

    1993-07-01

    The word autologous is Greek in origin. The definition is exact 'autos' means self and 'logus' means relation. Thus, the meaning is 'related to self'. Autologous blood transfusion, which also is referred to frequently but incorrectly and imprecisely as auto transfusion, designates the reinfusion of blood or blood components to the same individual from whom they were taken. Homologous blood is blood or blood components, from another human donor, taken and stored for later transfusion as required.

  17. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

    PubMed Central

    2011-01-01

    Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification. PMID:21244687

  18. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  19. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  20. Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts

    USDA-ARS?s Scientific Manuscript database

    Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full-length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed ...

  1. DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

    PubMed Central

    DiFranco, Marino; Quinonez, Marbella; Capote, Joana; Vergara, Julio

    2009-01-01

    A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the

  2. DNA transfection of mammalian skeletal muscles using in vivo electroporation.

    PubMed

    DiFranco, Marino; Quinonez, Marbella; Capote, Joana; Vergara, Julio

    2009-10-19

    A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver, skeletal muscle, and even the brain. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP, calpain, FKBP12, beta2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and alpha-actinin; and membrane proteins, i.e. alpha1s-DHPR, RyR1, cardiac Na/Ca(2+) exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical and functional evidence. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the

  3. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  4. Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

    PubMed Central

    Stroh, Thorsten; Erben, Ulrike; Kühl, Anja A.; Zeitz, Martin; Siegmund, Britta

    2010-01-01

    The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect. PMID:20209146

  5. CEA and AFP expression in human hepatoma cells transfected with antisense IGF-I gene

    PubMed Central

    Zhang, Li; Li, Shu-Nong; Wang, Xiao-Ning

    1998-01-01

    AIM: To determine whether antisense insulin-like growth factor-I (IGF-I) gene can modulate CEA and AFP expression in human hepatoma cells (HepG2). METHODS: Transfection of HepG2 cells was accomplished using Lipofectin reagent. Northern blot analysis confirmed the antisense IGF-I RNA of the transfected cells. CEA and AFP levels were measured using radioimmunoassay. RESULTS: Human hepatoma cell lines (HepG2) were transfected with antisense IGF-I gene. Northern blot analysis confirmed that antisense IGF-I RNA was expressed in the transfected cells. The effect of antisense IGF-I gene on CEA and AFP expression was demonstrated by the fact that the CEA and AFP levels in the supernatant of transfected cell culture were significantly lower as compared with the parent cells, [CEA 7.0 μg/L ± 0.76 μg/L and 3.29 μg/L ± 1.80 μg/L (P < 0.05) and AFP 53.63 μg/L ± 6.02 μg/L and 9.0 μg/L ± 5.26 μg/L (P < 0.01), respectively]. CONCLUSION: The malignant potentiality of the transfected cells was partially suppressed.Antisense IGF-I gene can modulate the expression of CEA and AFP in human hepatoma cell lines (HepG2) PMID:11819225

  6. Rebooting autoimmunity with autologous HSCT.

    PubMed

    Snowden, John A

    2016-01-07

    Autologous hematopoietic stem cell transplantation (HSCT) is increasingly used for severe autoimmune and inflammatory diseases, but the mechanisms involved have yet to be elucidated. In this issue of Blood, Delemarre et al report their findings in both animal and human models which provide insights into restoration of functionality and diversity within the regulatory T-cell (Treg) compartment following HSCT.

  7. Mechanistic investigations and molecular medicine applications of gold nanoparticle mediated (GNOME) laser transfection

    NASA Astrophysics Data System (ADS)

    Schomaker, M.; Heinemann, D.; Kalies, S.; Willenbrock, S.; Murua Escobar, H.; Buch, A.; Sodeik, B.; Ripken, T.; Meyer, H.

    2014-03-01

    Alternative high throughput transfection methods are required to understand the molecular network of the cell, which is linked to the evaluation of target genes as therapeutic agents. Besides diagnostic purposes, the transfection of primary- and stem cells is of high interest for therapeutic use. Here, the cell release of trans- or exogene proteins is used to develop immune cancer therapies. The basic requirement to accomplish manipulation of cells is an efficient and gentle transfection method. Therefore, we developed an automatized cell manipulation platform providing high throughput by using GNOME laser transfection. Herein, the interaction of moderately focused laser pulses with gold nanoparticles in close vicinity to the cell membrane mediate transient membrane permeabilization. The exact nature of the involved permeabilization effects depends on the applied particles and laser parameters. Hereinafter, we describe investigations considering the parameter regime, the permeabilization mechanism and the safety profile of GNOME laser transfection. The experimental and calculated results imply a combined permeabilization mechanism consisting of both photochemical and photothermal effects. Furthermore, paramount spatial control achieved either by laser illumination with micrometer precision or targeted gold nanoparticle binding to the cells was demonstrated, allowing selective cell manipulation and destruction. Additionally, the possibility to manipulate difficult to transfect primary cells (neurons) is shown. These results give insights in the basic mechanisms involved in GNOME laser transfection and serve as a strong basis to deliver different molecules for therapeutic (e.g. proteins) and diagnostic (siRNA) use.

  8. CMRF-56(+) blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses.

    PubMed

    Fromm, Phillip D; Papadimitrious, Michael S; Hsu, Jennifer L; Van Kooten Losio, Nicolas; Verma, Nirupama D; Lo, Tsun Ho; Silveira, Pablo A; Bryant, Christian E; Turtle, Cameron J; Prue, Rebecca L; Vukovic, Peter; Munster, David J; Nagasaki, Tomoko; Barnard, Ross T; Mahler, Stephen M; Anguille, Sébastien A; Berneman, Zwi; Horvath, Lisa G; Bradstock, Kenneth F; Joshua, Douglas E; Clark, Georgina J; Hart, Derek N J

    2016-06-01

    There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56(+) BDC, we showed that transfection of hCMRF-56(+) BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56(+) BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56(+) BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8(+) T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56(+) BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56(+) BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

  9. Transfection-mediated recombination of influenza A virus.

    PubMed Central

    Bergmann, M; García-Sastre, A; Palese, P

    1992-01-01

    Several mechanisms, including a high mutation rate and reassortment of genes, have been found to be responsible for the variability of influenza A viruses. RNA recombination would be another mechanism leading to genetic variation; however, recombination has only rarely been reported to occur in influenza viruses. During ribonucleoprotein transfection experiments designed to generate viable influenza viruses from in vitro-synthesized RNA, we discovered several viruses which must have originated from recombination events. The ribonucleoprotein transfection system may enhance the formation of viruses which result from jumping of the viral polymerase between RNAs or from ligation of different viral RNAs. Five different recombinant viruses are described. Two of these, REC1 and REC2, contain a neuraminidase (NA) gene whose defective polyadenylation signal has been repaired via intergenic recombination; 124 and 95 nucleotides have been added, respectively. Another virus, REC5, must have originated by multiple recombination events since it contains a mosaic gene with sequences derived from the NA gene of influenza A/WSN/33 virus and the matrix, polymerase protein PB1, and NA genes of influenza A/PR/8/34 virus. Images PMID:1279208

  10. Chromatin-binding regions of EBNA1 protein facilitate the enhanced transfection of Epstein-Barr virus-based vectors.

    PubMed

    Howden, Sara E; Wardan, Hady; Voullaire, Lucille; McLenachan, Samuel; Williamson, Robert; Ioannou, Panos; Vadolas, Jim

    2006-08-01

    Epstein-Barr virus (EBV)-based vectors can stably maintain large genomic fragments in mammalian cells, offering great potential for the treatment/correction of many acquired and inherited disorders. Numerous studies report marked increases in the transfection efficiency of EBV-based vectors after delivery into cell lines constitutively expressing Epstein-Barr nuclear antigen-1 (EBNA1), compared with cells not expressing EBNA1. We employ a novel strategy, involving the transfection of mRNA encoding EBNA1, to transiently express EBNA1 protein in human cells. Subsequently we show that the transfection efficiency of a 21-kb EBVbased vector is improved significantly when codelivered with mRNA encoding EBNA1. Similar increases in transfection efficiency were observed after delivery of the plasmid into cells constitutively expressing EBNA1. We also investigate the mechanism by which EBNA1 facilitates the transfection of EBV-based vectors, using mRNA encoding modified versions of the protein. Previous studies suggest that the EBNA1 DNA-binding domain (DBD), together with the nuclear localization signal (NLS), may enhance transfection of EBV plasmids by facilitating their nuclear transport. We demonstrate that an EBNA1 derivative comprising only the NLS and DBD does not facilitate transfection of EBV-based vectors. However, cells expressing an EBNA1 derivative devoid of a functional NLS but retaining the chromatin-binding regions, domains A and B, enhances plasmid transfection efficiency by up to 10-fold. Moreover, a variant of EBNA1 comprising two copies of domain A fused to the DBD enhances DNA transfection to an even greater extent than wild-type EBNA1. We therefore propose that EBNA1-mediated transfection of EBV-based vectors is dependent on the presence of chromatin- binding regions and the DBD, but not the NLS.

  11. Development of human granulocyte-macrophage colony-stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer.

    PubMed

    Hogge, G S; Burkholder, J K; Culp, J; Albertini, M R; Dubielzig, R R; Keller, E T; Yang, N S; MacEwen, E G

    1998-09-01

    Cytokine gene-engineered tumor vaccines are currently an area of intense investigation in both basic research and clinical medicine. Our efforts to utilize tumor vaccines in an immunotherapeutic manner involve canines with spontaneous tumors. We hypothesized that canine tumor cells, transfected with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA in a plasmid vector, would prove nontoxic following intradermal administration, generate biologically relevant levels of protein, effect local histological changes at the sites of vaccination, and create a systemic antitumor response. Sixteen tumor-bearing dogs were admitted to a study of ex vivo gene therapy. Tumor tissue was surgically removed, enzymatically and mechanically dissociated, irradiated, transfected, and intradermally injected back into the patients. The dogs were vaccinated with primary autologous tumor cells transfected with hGM-CSF or a reporter control gene. hGM-CSF protein was detected (0.07 to 14.15 ng/vaccination site) at 24 hr postinjection and dramatic histological changes were observed, characterized by neutrophil and macrophage infiltration at the sites of injection of hGM-CSF-transfected tumor cells. This was in stark contrast to the lesser neutrophilic and eosinophilic infiltrates found at control vaccination sites. Objective evidence of an antitumor response was observed in three animals. These data, in a large animal translational model of spontaneous tumors, demonstrate in vivo biological activity of hGM-CSF-transfected autologous tumor cell vaccines.

  12. Autologous umbilical cord blood transfusion.

    PubMed Central

    Ballin, A.; Arbel, E.; Kenet, G.; Berar, M.; Kohelet, D.; Tanay, A.; Zakut, H.; Meytes, D.

    1995-01-01

    The purpose of this study was to examine some aspects of umbilical cord blood collection for autologous transfusion in premature infants. All 120 microbacterial cultures (aerobic and anaerobic) of cord blood samples as well as 30 cultures of mycoplasma were treated. Cord prothrombin fragment (F 1 + 2) concentrations were quantified at one and 10 minutes after clamping of the cord. F 1 + 2 concentrations assessed on 25 newborn infants were similar and no linear association with time of clamping could be drawn. This means that cord blood thrombosis is not activated for at least 10 minutes following clamping of the cord. As far as is known, the first newborn infant to benefit from this method of transfusion is reported here. The premature infant received two portions of autologous blood (on days 5 and 7). No untoward effects were noted. Blood, collected from the umbilical cord, is a safe source for autotransfusion, provided that bacteriological testing has been carried out. PMID:8535878

  13. Rhinoplasty using autologous costal cartilage.

    PubMed

    Miranda, Nancy; Larocca, Carlos Gil; Aponte, Ciro

    2013-06-01

    Most Latin American patients looking to have a primary septorhinoplasty share common characteristics in relation to an incorrect projection of the nasal tip complex and a low dorsal line. Thus, the frequent use of structural techniques and of surgical enhancement techniques becomes necessary to improve the nasal contour. In cases of secondary septorhinoplasty, it is also usual in our practice not to have sufficient septal cartilage available or with the required quality to give structure and support to the nasal tip complex, handle the nasal dorsum, and simultaneously correct postseptorhinoplasty deformities. For these reasons, in our practice costal cartilage represents an excellent option as autologous graft material. We present our experience using autologous costal cartilage for structural and nonstructural purposes in 286 selected patients who underwent open rhinoplasty between 2004 and 2011. We emphasize preoperative analyses, we discuss the criteria for selecting costal graft as graft material, we show key aspects of the dynamic of the surgery, and we consider the possibility of using autologous costal graft in combination with heterologous grafts. In this work we also establish the disadvantages of costal cartilage as graft material in specific areas of the surgical anatomy of the nose.

  14. Proliferation and Differentiation of Rat Osteoporosis Mesenchymal Stem Cells (MSCs) after Telomerase Reverse Transcriptase (TERT) Transfection

    PubMed Central

    Li, Chao; Wei, Guojun; Gu, Qun; Wang, Qiang; Tao, Shuqin; Xu, Liang

    2015-01-01

    Background The aim of this study was to determine whether MSC are excellent materials for MSCs transplantation in the treatment of osteoporosis. Material/Methods We studied normal, osteoporosis, and TERT-transfected MSC from normal and osteoporosis rats to compare the proliferation and osteogenic differentiation using RT-PCR and Western blot by constructing an ovariectomized rat model of osteoporosis (OVX). The primary MSC from model rats were extracted and cultured to evaluate the proliferation and differentiation characteristics. Results MSCs of osteoporosis rats obviously decreased in proliferation ability and osteogenic differentiation compared to that of normal rats. In contrast, in TERT-transfected MSC, the proliferation and differentiation ability, and especially the ability of osteogenic differentiation, were significantly higher than in osteoporosis MSC. Conclusions TERT-transfected MSCs can help osteoporosis patients in whom MSC proliferation and osteogenic differentiation ability are weak, with an increase in both bone mass and bone density, becoming an effective material for autologous transplantation of MSCs in further treatment of osteoporosis. However, studies are still needed to prove the in vivo effect, biological safety, and molecular mechanism of TERT-osteoporosis treatment. Additionally, because the results are from an animal model, more research is needed in generalizing rat model findings to human osteoporosis patients. PMID:25796354

  15. Transfectability of rough strains of Salmonella typhimurium.

    PubMed Central

    Bursztyn, H; Sgaramella, V; Ciferri, O; Lederberg, J

    1975-01-01

    Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl2. The yield of infectious centers is about 10(-8) per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate-galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are not longer competent for transfection. PMID:1104596

  16. Optimizacion of Babesia bovis transfection methods

    USDA-ARS?s Scientific Manuscript database

    The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In thi...

  17. Ex vivo production of autologous whole inactivated HIV-1 for clinical use in therapeutic vaccines.

    PubMed

    Gil, Cristina; Climent, Núria; García, Felipe; Hurtado, Carmen; Nieto-Márquez, Sara; León, Agathe; García, M Teresa; Rovira, Cristina; Miralles, Laia; Dalmau, Judith; Pumarola, Tomás; Almela, Manel; Martinez-Picado, Javier; Lifson, Jeffrey D; Zamora, Laura; Miró, José M; Brander, Christian; Clotet, Bonaventura; Gallart, Teresa; Gatell, José M

    2011-08-05

    This study provides a detailed description and characterization of the preparation of individualized lots of autologous heat inactivated HIV-1 virions used as immunogen in a clinical trial designed to test an autologous dendritic-cell-based therapeutic HIV-1 vaccine (Clinical Trial DCV-2, NCT00402142). For each participant, ex vivo isolation and expansion of primary virus were performed by co-culturing CD4-enriched PBMCs from the HIV-1-infected patient with PBMC from HIV-seronegative unrelated healthy volunteer donors. The viral supernatants were heat-inactivated and concentrated to obtain 1 mL of autologous immunogen, which was used to load autologous dendritic cells of each patient. High sequence homology was found between the inactivated virus immunogen and the HIV-1 circulating in plasma at the time of HIV-1 isolation. Immunogens contained up to 10⁹ HIV-1 RNA copies/mL showed considerably reduced infectivity after heat inactivation (median of 5.6 log₁₀), and were free of specified adventitious agents. The production of individualized lots of immunogen based on autologous inactivated HIV-1 virus fulfilling clinical-grade good manufacturing practice proved to be feasible, consistent with predetermined specifications, and safe for use in a clinical trial designed to test autologous dendritic cell-based therapeutic HIV-1 vaccine.

  18. Preeclampsia in autologous and oocyte donation pregnancy: is there a different pathophysiology?

    PubMed

    Lashley, Lisa E E L O; Buurma, Aletta; Swings, Godelieve M J S; Eikmans, Michael; Anholts, Jacqueline D H; Bakker, Jaap A; Claas, Frans H J

    2015-06-01

    Oocyte donation (OD) is a specific method of artificial reproductive technology that is accompanied by a higher risk of preeclampsia during pregnancy. The pathophysiological mechanism underlying preeclampsia in OD pregnancies is thought to differ from preeclampsia in autologous pregnancies. As preeclampsia in autologous pregnancies is suggested to be associated with complement activation, we studied C4d deposition, circulating complement components and placental complement regulatory proteins in preeclamptic OD pregnancies. Women with uncomplicated and preeclamptic pregnancies after OD or spontaneous conception were selected. We stained the placentas for C4d, marker for complement activation, measured complement factors C1q, C3 and C4 in maternal sera and quantified the placental mRNA expression of complement regulatory proteins CD46, CD55 and CD59. A significantly (p < 0.03) higher incidence of C4d deposition was observed in placentas from women with preeclampsia compared with uncomplicated pregnancies, both OD and autologous. The level of complement factors in serum did not differ between the groups. Children born in the autologous preeclampsia group were significantly lower in birth weight (p < 10th percentile) compared with the preeclamptic OD group. In addition, the placental mRNA expression level of complement regulatory proteins was significantly lower in uncomplicated and preeclamptic OD compared with the autologous pregnancies. In line with autologous preeclampsia pregnancies, there is excessive activation of complement in preeclamptic OD pregnancies. However, in contrast to autologous pregnancies this is not associated with counterbalancing upregulation of complement regulatory proteins. Furthermore, C4d deposition in OD pregnancies is not related to the severity of preeclampsia, suggesting another trigger or regulatory mechanism of placental C4d deposition in preeclamptic OD pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    PubMed Central

    Sork, Helena; Nordin, Joel Z; Turunen, Janne J; Wiklander, Oscar PB; Bestas, Burcu; Zaghloul, Eman M; Margus, Helerin; Padari, Kärt; Duru, Adil D; Corso, Giulia; Bost, Jeremy; Vader, Pieter; Pooga, Margus; Smith, CI Edvard; Wood, Matthew JA; Schiffelers, Raymond M; Hällbrink, Mattias; Andaloussi, Samir EL

    2016-01-01

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents. PMID:27111416

  20. 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

  1. 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.

  2. Autologous Blood Transfusion in Sports: Emerging Biomarkers.

    PubMed

    Salamin, Olivier; De Angelis, Sara; Tissot, Jean-Daniel; Saugy, Martial; Leuenberger, Nicolas

    2016-07-01

    Despite being prohibited by the World Anti-Doping Agency, blood doping through erythropoietin injection or blood transfusion is frequently used by athletes to increase oxygen delivery to muscles and enhance performance. In contrast with allogeneic blood transfusion and erythropoietic stimulants, there is presently no direct method of detection for autologous blood transfusion (ABT) doping. Blood reinfusion is currently monitored with individual follow-up of hematological variables via the athlete biological passport, which requires further improvement. Microdosage is undetectable, and suspicious profiles in athletes are often attributed to exposure to altitude, heat stress, or illness. Additional indirect biomarkers may increase the sensitivity and specificity of the longitudinal approach. The emergence of "-omics" strategies provides new opportunities to discover biomarkers for the indirect detection of ABT. With the development of direct quantitative methods, transcriptomics based on microRNA or messenger RNA expression is a promising approach. Because blood donation and blood reinfusion alter iron metabolism, quantification of proteins involved in metal metabolism, such as hepcidin, may be applied in an "ironomics" strategy to improve the detection of ABT. As red blood cell (RBC) storage triggers changes in membrane proteins, proteomic methods have the potential to identify the presence of stored RBCs in blood. Alternatively, urine matrix can be used for the quantification of the plasticizer di(2-ethyhexyl)phthalate and its metabolites that originate from blood storage bags, suggesting recent blood transfusion, and have an important degree of sensitivity and specificity. This review proposes that various indirect biomarkers should be applied in combination with mathematical approaches for longitudinal monitoring aimed at improving ABT detection. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Modulation of MUC1 mucin as an escape mechanism of breast cancer cells from autologous cytotoxic T-lymphocytes

    PubMed Central

    Kontani, K; Taguchi, O; Narita, T; Izawa, M; Hiraiwa, N; Zenita, K; Takeuchi, T; Murai, H; Miura, S; Kannagi, R

    2001-01-01

    MUC1 mucin is known to serve as a target molecule in the killing of breast cancer cells by cytotoxic T-lymphocytes (CTLs). We searched for a possible mechanism allowing tumour cells to escape from autologous CTLs. When the killing of breast cancer cells by autologous lymphocytes was examined in 26 patients with breast cancer, significant tumour cell lysis was observed in 8 patients, whereas virtually no autologous tumour cell lysis was detected in as many as 18 patients. In the patients who showed negligible tumour cell lysis, the autologous tumour cells expressed MUC1-related antigenic epitopes much more weakly than the tumour cells in the patients who exhibited strong cytotoxicity (significant statistically at P< 0.0005–0.0045), suggesting that the unresponsiveness of cancer cells to CTLs observed in these patients was mainly due to loss of MUC1 expression or modulation of its antigenicity. A breast cancer cell line, NZK-1, established from one of the cytotoxicity-negative patients, did not express MUC1 and was resistant to killing by CTLs, while control breast cancer cell lines expressing MUC-1 were readily killed by CTLs. Transfection of NZK-1 cells with MUC1 cDNA induced significant lysis by autologous T-lymphocytes. These results supported the importance of MUC1 mucin in autologous anti-tumour immunity, but suggested that the major escape mechanism of tumour cells from autologous T-lymphocytes is the loss and/or modulation of MUC1 antigenicity on tumour cells, which would limit the effectiveness of possible immunotherapy designed to target the MUC1 mucin. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336479

  4. Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene.

    PubMed

    Ning, Bo-Tao; Tang, Yong-Min

    2012-04-01

    CD14 is the pivotal molecule in the diagnosis and therapy of CD14-associated diseases, and is important in bacteremia. The HeLa cell line is regarded as immortal due to its prolific character. The HeLa cell line is derived from human cervical cancer cells and has been widely used in cancer research and gene transfection. In the present study, we established the expression plasmid pcDNA3.1(+)-CD14, and transfected it into the human cervical cancer cell line HeLa to establish a stable cell line (HeLa-CD14) expressing human CD14 antigen on the membrane. After the human CD14 gene was cloned and sequenced through RT-PCR and T-A cloning techniques, the eukaryotic expression vector pcDNA3.1(+)-CD14 was constructed by cleaving with double restriction endonucleases and ligating with T4 ligase. HeLa cells were transfected with the pcDNA3.1(+)-CD14 recombinant plasmid using Superfect transfection reagent. The cells were selected using G418 and the expression of human CD14 on the transfectant was confirmed by RT-PCR and immunohistochemistry. The expression of CD14 mRNA was significantly different between the blank pcDNA3.1(+)-transfected cell group and the pcDNA3.1(+)-CD14-transfected cell group (p<0.01). The fluorescence was significantly stronger on the established stable cell line than on the transiently transfected HeLa cells, and no visible fluorescence was observed in blank pcDNA3.1(+)-transfected cells. In this study, the human CD14 transfectant, stable cell line HeLa-CD14, was successfully established, which may be used to study CD14 and cervical cancer in vitro and in vivo.

  5. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

    PubMed Central

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

    2016-01-01

    This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

  6. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    NASA Astrophysics Data System (ADS)

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-04-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

  7. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    PubMed Central

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-01-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired. PMID:27067121

  8. Simplified enrollment for autologous transfusion: automatic referral of presurgical patients for assessment for autologous blood collections.

    PubMed

    Moore, S B; Swenke, P K; Foss, M L; Rand, J A; Cabanela, M E; Kavanagh, B; Taswell, H F

    1992-04-01

    We implemented a pilot program at our institution for automatic referral of patients for presurgical assessment for preoperative and intraoperative collection of autologous blood. Although patients and clinicians support the use of autologous transfusion, often a request for collection of autologous blood is not initiated. During 11 months, 269 patients (82%) of three orthopedic surgeons entered the program, and 218 underwent operation and were dismissed from the hospital. A total of 940 units of autologous blood (675 preoperatively and 265 intraoperatively) was collected from these 218 patients, and 84% of the units were transfused. Throughout hospitalization, 86% of the patients received only autologous blood, whereas 14% received various proportions of homologous and autologous blood. In contrast, only 26% of a concomitant control group of 220 consecutive orthopedic surgical patients not participating in the automatic-referral program received only autologous blood. Thus, the automatic-referral program increased the percentage of elective orthopedic surgical patients who received only autologous blood from 26% to 86% (P less than 0.001). This study also showed that the same amount of blood was used for autologous transfusions as was routinely used for homologous transfusions in similar cases. The automatic-referral system was convenient for physicians and patients and offered the benefits of reduction of transfusion-associated risks and amelioration of patient anxieties.

  9. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    NASA Astrophysics Data System (ADS)

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-12-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.

  10. Size specific transfection to mammalian cells by micropillar array electroporation

    NASA Astrophysics Data System (ADS)

    Zu, Yingbo

    Electroporation serves as a promising non-viral gene delivery approach, while its current configurations carry drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here, we developed a new micropillar array electroporation (MAE) platform to advance the delivery of plasmid DNA and RNA to mammalian cells. By introducing well-patterned micropillar array on the electrode surface, the number of pillars each cell faces varies with its cell membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently done and contributed to a 2.5 3 fold increase on plasmid DNA transfection and an additional 10-55% knockdown with targeting siRNA, respectively. The delivery efficiency varies with the number and size of the micropillars as well as their pattern density. As MAE works like many single cell electroporation is carried out in a parallel fashion, the electrophysiology response of individual cells is representative, which has the potential to gear up the tedious, cell-specific protocol screening process in the current in vitro bulk electroporation (i.e., electroporation to a large population of cells). Its success might facilitate the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.

  11. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-01-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments. PMID:27924861

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

    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.

  13. 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.

  14. Characterization of CD8+ T-cell responses in the peripheral blood and skin injection sites of melanoma patients treated with mRNA electroporated autologous dendritic cells (TriMixDC-MEL).

    PubMed

    Benteyn, Daphné; Van Nuffel, An M T; Wilgenhof, Sofie; Corthals, Jurgen; Heirman, Carlo; Neyns, Bart; Thielemans, Kris; Bonehill, Aude

    2013-01-01

    Treatment of melanoma patients with mRNA electroporated dendritic cells (TriMixDC-MEL) stimulates T-cell responses against the presented tumor-associated antigens (TAAs). In the current clinical trials, melanoma patients with systemic metastases are treated, requiring priming and/or expansion of preexisting TAA-specific T cells that are able to migrate to both the skin and internal organs. We monitored the presence of TAA-specific CD8(+) T cells infiltrating the skin at sites of intradermal TriMixDC-MEL injection (SKILs) and within the circulation of melanoma patients treated in two clinical trials. In 10 out of fourteen (71%) patients screened, CD8(+) T cells recognizing any of the four TAA presented by TriMixDC-MEL cellular vaccine were found in both compartments. In total, 30 TAA-specific T-cell responses were detected among the SKILs and 29 among peripheral blood T cells, of which 24 in common. A detailed characterization of the antigen specificity of CD8(+) T-cell populations in four patients indicates that the majority of the epitopes detected were only recognized by CD8(+) T cells derived from either skin biopsies or peripheral blood, indicating that some compartmentalization occurs after TriMix-DC therapy. To conclude, functional TAA-specific CD8(+) T cells distribute both to the skin and peripheral blood of patients after TriMixDC-MEL therapy.

  15. Isolation, culture, and transfection of melanocytes.

    PubMed

    Godwin, Lauren S; Castle, Joanna T; Kohli, Jaskaren S; Goff, Philip S; Cairney, Claire J; Keith, W Nicol; Sviderskaya, Elena V; Bennett, Dorothy C

    2014-06-03

    Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal-like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells.

  16. 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

  17. Optimization of conditions for transfection with the Sofast gene vector.

    PubMed

    Zhou, Lei; Liu, Fan; Qiao, Fang-Fang; Tong, Man-Li; Fu, Zuo-Gen; Dan, Bing; Yang, Tian-Ci; Zhang, Zhong-Ying

    2011-01-01

    We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

  18. Transfection of C6 Glioma Cells with Connexin 43 cDNA: Analysis of Expression, Intercellular Coupling, and Cell Proliferation

    NASA Astrophysics Data System (ADS)

    Zhu, D.; Caveney, S.; Kidder, G. M.; Naus, C. C. G.

    1991-03-01

    C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than nontransfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

  19. Mussel Adhesion-Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose-Derived Stem Cells.

    PubMed

    Shin, Jisoo; Cho, Jung Ho; Jin, Yoonhee; Yang, Kisuk; Lee, Jong Seung; Park, Hyun-Ji; Han, Hyung-Seop; Lee, Jinkyu; Jeon, Hojeong; Shin, Heungsoo; Cho, Seung-Woo

    2016-12-01

    Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution-mediated transfection are limited due to low transfection efficiency and insufficient duration of cell-siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio-inspired polymer-mediated reverse transfection system is developed consisting of implantable poly(lactic-co-glycolic acid) (PLGA) scaffolds functionalized with siRNA-lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA-coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA-sLNP-PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose-derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA-sLNP-PLGA scaffolds enhanced bone formation in a mouse model of critical-sized bone defect. Therefore, the bio-inspired reverse transfection system can provide an all-in-one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Subcellular localization of RNAs in transfected cells: role of sequences at the 5' terminus.

    PubMed Central

    Alfonso-Pizarro, A; Carlson, D P; Ross, J

    1984-01-01

    We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported. Images PMID:6209612

  1. Evolutionarily stable anti-cancer therapies by autologous cell defection.

    PubMed

    Archetti, Marco

    2013-01-01

    Game theory suggests an anti-cancer treatment based on the use of modified cancer cells that disrupt cooperation within the tumor. Cancer cells are harvested from the patient, the genes for the production of essential growth factors are knocked out in vitro and the cells are then reinserted in the tumor, where they lead to its collapse. Current anti-cancer drugs and treatments based on gene therapy are prone to the evolution of resistance, because cancer is a process of clonal selection: resistant cell lines have a selective advantage and therefore increase in frequency, eventually conferring resistance to the whole tumor and leading to relapse. An effective treatment must be evolutionarily stable, that is, immune to the invasion of resistant mutant cells. This study shows how such a treatment can be achieved by autologous cell therapy using modified cancer cells, knocked out for genes coding for diffusible factors like growth factors. The evolutionary dynamics of a population of cells producing diffusible factors are analyzed using a nonlinear public goods game in a structured population in which the interaction neighborhood and the update neighborhood are decoupled. The analysis of the dynamics of the system reveals what interventions can drive the population to a stable equilibrium in which no diffusible factors are produced. A treatment based on autologous knockout cell therapy can be designed to lead to the spontaneous collapse of a tumor, without targeting directly the cancer cells, their growth factors or their receptors. Critical parameters that can make the therapy effective are identified. Concepts from evolutionary game theory and mechanism design, some of which are counterintuitive, can be adopted to optimize the treatment. Although it shares similarities with other approaches based on gene therapy and RNA interference, the method suggested here is evolutionarily stable under certain conditions. This method, named autologous cell defection, can be

  2. Evolutionarily stable anti-cancer therapies by autologous cell defection

    PubMed Central

    Archetti, Marco

    2013-01-01

    Game theory suggests an anti-cancer treatment based on the use of modified cancer cells that disrupt cooperation within the tumor. Cancer cells are harvested from the patient, the genes for the production of essential growth factors are knocked out in vitro and the cells are then reinserted in the tumor, where they lead to its collapse. Background and objectives: Current anti-cancer drugs and treatments based on gene therapy are prone to the evolution of resistance, because cancer is a process of clonal selection: resistant cell lines have a selective advantage and therefore increase in frequency, eventually conferring resistance to the whole tumor and leading to relapse. An effective treatment must be evolutionarily stable, that is, immune to the invasion of resistant mutant cells. This study shows how such a treatment can be achieved by autologous cell therapy using modified cancer cells, knocked out for genes coding for diffusible factors like growth factors. Methodology: The evolutionary dynamics of a population of cells producing diffusible factors are analyzed using a nonlinear public goods game in a structured population in which the interaction neighborhood and the update neighborhood are decoupled. The analysis of the dynamics of the system reveals what interventions can drive the population to a stable equilibrium in which no diffusible factors are produced. Results: A treatment based on autologous knockout cell therapy can be designed to lead to the spontaneous collapse of a tumor, without targeting directly the cancer cells, their growth factors or their receptors. Critical parameters that can make the therapy effective are identified. Concepts from evolutionary game theory and mechanism design, some of which are counterintuitive, can be adopted to optimize the treatment. Conclusions and implications: Although it shares similarities with other approaches based on gene therapy and RNA interference, the method suggested here is evolutionarily stable under

  3. Plasma-mediated transfection of RPE

    NASA Astrophysics Data System (ADS)

    Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

    2006-02-01

    A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

  4. [Autologous fat grafting and rhinoplasty].

    PubMed

    Nguyen, P S; Baptista, C; Casanova, D; Bardot, J; Magalon, G

    2014-12-01

    Revision rhinoplasty can be very challenging especially in cases of thin skin. Autologous fat graft is utilized in numerous applications in plastic surgery; however, its use relative to the nasal region remains uncommon. Adipose tissue, by virtue of its volumetric qualities and its action on skin trophicity, can be considered to be a gold standard implant. From 2006 until 2012, we have treated patients by lipofilling in order to correct sequelae of rhinoplasty. The mean quantity of adipose tissue injected was 2.1cm(3) depending on the importance of the deformity and the area of injection: irregularity of the nasal dorsum, visible lateral osteotomies, saddle nose. Following the course of our practice, we conceived micro-cannulas that allow a much greater accuracy in the placement of the graft and enable to perform interventions under local anesthesia. These non-traumatic micro-cannulas do not cause post-operative ecchymosis and swelling which shorten the recovery time for the patient. On patients who have undergone multiple operations, lipofilling can be a simple and reliable alternative to correct imperfections that may take place after a rhinoplasty.

  5. Improved fat transplantation survival by using the conditioned medium of vascular endothelial growth factor transfected human adipose-derived stem cells.

    PubMed

    Zhang, Yang; Xiao, Li-Ling; Li, Jiang-Xuan; Liu, Hong-Wei; Li, Sheng-Hong; Wu, Yan-Yun; Liao, Xuan; Rao, Cong-Qiang

    2017-08-01

    Autologous fat transplantation has been applied widely in clinic. However, the low survival rate is still a problem to be solved. Studies shows that the human adipose-derived stem cells (ADSCs) transfected by vascular endothelial growth factor (VEGF) can improve the survival rate of autologous fat transplantation. Our study is to evaluate the effects of the conditioned medium of VEGF-transfected human adipose-derived stem cells (VEGF-ADSCs-CM) on fat transplantation. ADSCs were isolated and transfected with MOI = 40. The study was divided into three groups, VEGF-ADSCs-CM group, normal-ADSCs-CM group and control group. The conditioned media for VEGF-ADSCs-CM group and normal-ADSCs-CM group were collected, and then mixed with fat, with the mixtures being injected into the back of nude mice. On 4, 7, 15, 30, 60 days after transplantation, the grafts were evaluated on the wet weight, histology, ELISA and western blot. As the results revealed, the survival rate of VEGF-ADSCs-CM group was highest with the best fat cell morphology, and the VEGF secretion of VEGF-ADSCs-CM group was also highest. Therefore, our study demonstrates that VEGF-ADSCs-CM can improve the survival rate of fat transplantation effectively, and VEGF-ADSCs-CM can be regarded as an effective assisted method for fat transplantation. Copyright © 2017 Kaohsiung Medical University. Published by Elsevier Taiwan. All rights reserved.

  6. Transfection of NF-κB decoy oligodeoxynucleotide suppresses pulmonary metastasis by murine osteosarcoma.

    PubMed

    Nishimura, A; Akeda, K; Matsubara, T; Kusuzaki, K; Matsumine, A; Masuda, K; Gemba, T; Uchida, A; Sudo, A

    2011-04-01

    Nuclear factor-kappa B (NF-κB) has a pivotal role in the progression and distant metastasis of cancers, including malignant bone tumors. To inhibit NF-κB activation, a new molecular therapy using synthetic double-stranded oligodeoxynucleotide (ODN) as a 'decoy' cis element against NF-κB has been developed. To determine whether pulmonary metastasis of osteosarcoma is reduced by inhibiting the action of NF-κB, NF-κB decoy ODN was transfected into the nuclei of murine osteosarcoma cells with high pulmonary metastatic potential, the LM8 cell line, using a three-dimensional alginate spheroid culture model. An in vitro study demonstrated the successful transfection of LM8 cells cultured in alginate beads by 'naked' NF-κB decoy ODN and that the activation of NF-κB signaling was significantly suppressed. Tumor growth was not affected by transfection of NF-κB decoy ODN, however, the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) mRNA was markedly decreased. Furthermore, the transfection of 'naked' NF-κB decoy ODN effectively suppressed pulmonary metastasis in an in vivo alginate bead transplantation model. Our results suggest that NF-κB has a central and specific role in the regulation of tumor metastasis and could be a molecular target for development of anti-metastatic treatments for osteosarcoma.

  7. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

    PubMed

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-05-11

    Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

  8. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

    PubMed Central

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-01-01

    Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors. PMID:27165510

  9. 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

  10. Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

    PubMed Central

    Nii, Takenobu; Kohara, Hiroshi; Marumoto, Tomotoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Tani, Kenzaburo

    2016-01-01

    Abstract Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell state were transfected using nonliposomal transfection reagents with plasmid DNA or mRNA encoding enhanced green fluorescent protein. We found that most cells (DNA > 90%; mRNA > 99%) were transfected without the loss of undifferentiated PSC marker expression or pluripotency. Moreover, we demonstrated an efficient gene editing method using transcription activator-like effector nucleases (TALENs) targeting the adenomatous polyposis coli (APC) gene. Our new method may improve hPSC gene transfer techniques, thus facilitating their use for human regenerative medicine. PMID:27257519

  11. [Establishment of murine cell line transfected with human CD14 gene].

    PubMed

    Ning, Bo-Tao; Tang, Yong-Min; Xu, Yan; Chen, Yan-Fei; Cao, Jiang

    2006-04-01

    This study was aimed to construct the CD14 eukaryotic expression vector, establish the transgeneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M(5)). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone techniques. Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases and ligating with T4 ligase. A murine melanoma cell line B16 was transfected with the pcDNA3.1(+)/CD14 recombinant with Superfect transfection reagent. Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry (FCM). The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank database. The recombinant pcDNA3.1(+)/CD14 was identified with double-enzyme cleaving. The expression of the human CD14 on the transfectant (B16/CD14) was confirmed by FCM. In conclusion, the murine cell line B16/CD14 fransfected with human CD14 gene has been established which can be used for the study of human AML-M(5) antibody targeting therapy with mouse model.

  12. Autologous stem cell transplant with gene therapy for Friedreich ataxia.

    PubMed

    Tajiri, Naoki; Staples, Meaghan; Kaneko, Yuji; Kim, Seung U; Zesiewicz, Theresa A; Borlongan, Cesar V

    2014-09-01

    We advance the overarching hypothesis that stem cell therapy is a potent treatment for Friedreich's ataxia (FRDA). Here, we discuss the feasibility of autologous transplantation in FRDA, highlighting the need for the successful isolation of the FRDA patient's bone marrow-derived mesenchymal stem cells, followed by characterization that these cells maintain the GAA repeat expansion and the reduced FXN mRNA expression, both hallmark features of FRDA. Next, we discuss the need for assessment of the proliferative capability and pluripotency of FRDA patient's bone marrow-derived mesenchymal stem cells. In particular, we view the need for characterizing the in vitro differentiation of bone marrow-derived mesenchymal stem cells into the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. Finally, we discuss the need to test the application of bone marrow-derived mesenchymal stem cells as potent autologous donor cells for FRDA. The demonstration of the functional correction of the mutated gene in these cells will be a critical endpoint of determining the potential of stem cell therapy in FRDA. We envision a gene-based cell transplant strategy as a likely therapeutic approach for FRDA, involving stable insertion of functional human bacterial artificial chromosomes or BACs containing the intact FXN gene into stem cells, thereafter leading to the expression of frataxin protein in differentiated neurons/cardiomyocytes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent.

    PubMed

    Guo, Xuan-Yan; Lu, Man; Chen, Xue-Qing; He, Fan-Ding; Li, Ang

    2016-06-01

    To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells. Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively. The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = -4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05). A549 cells transfected by ultrasound microbubble

  14. Transfection of mammalian cells using block copolypeptide vesicles.

    PubMed

    Sun, Victor Z; Choe, Uh-Joo; Rodriguez, April R; Dai, Howard; Deming, Timothy J; Kamei, Daniel T

    2013-05-01

    An arginine-leucine block copolypeptide (R60 L20 ) is synthesized, which is capable of forming vesicles with controllable sizes, able to transport hydrophilic cargo across the cell membrane, and exhibit relatively low cytotoxicity. The R60 L20 vesicles also possess the ability to deliver DNA into mammalian cells for transfection. Although the transfection efficiency is lower than that of the commercially available transfection agent Lipofectamine 2000, the R60 L20 vesicles are able to achieve transfection with significantly lower cytotoxicity and immunogenicity. This behavior is potentially due to its stronger interaction with DNA which subsequently provides better protection against anionic heparin.

  15. Transabdominal sacrocolpopexy with autologous rectus fascia graft.

    PubMed

    Abraham, Nitya; Quirouet, Adrienne; Goldman, Howard B

    2016-08-01

    Extrusion and infection are potential postoperative complications when using synthetic mesh for abdominal sacrocolpopexy. Long-term follow-up in the Colpopexy and Urinary Reduction Efforts (CARE) trial revealed an estimated 9.9 % risk of mesh extrusion. There are 26 reports of spondylodiscitis after sacrocolpopexy with synthetic mesh. These surgical risks may be decreased by using autologous fascia. To date, there have been no reports of extrusion or spondylodiscitis after using autologous fascia for sacrocolpopexy. This video demonstrates transabdominal sacrocolpopexy with an autologous rectus fascia graft. A 76-year-old woman with symptomatic stage 3 prolapse also had a history of diverticulitis and sigmoid abscess requiring sigmoid colectomy with end colostomy and incidental left ureteral transection with subsequent left nephrostomy tube placement. She presented for colostomy reversal, ureteral reimplantation, and prolapse repair. Given the need for concomitant colon and ureteral reconstruction, the risk of infection was potentially higher if synthetic mesh were used. The patient therefore underwent transabdominal sacrocolpopexy with autologous rectus fascia graft. At 4 months' follow-up the patient reported resolution of her symptoms and on examination she had no pelvic organ prolapse. Transabdominal sacrocolpopexy using autologous rectus fascia graft is a feasible option, especially in cases in which infection and synthetic mesh extrusion risks are potentially higher.

  16. Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

    PubMed Central

    Wang, Tongguang; Choi, Elliot; Monaco, Maria Chiara G.; Campanac, Emilie; Medynets, Marie; Do, Thao; Rao, Prashant; Johnson, Kory R.; Elkahloun, Abdel G.; Von Geldern, Gloria; Johnson, Tory; Subramaniam, Sriram; Hoffman, Dax; Major, Eugene; Nath, Avindra

    2013-01-01

    Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. PMID:24303066

  17. Coding potential of transfected human placental lactogen genes.

    PubMed Central

    Reséndez-Pérez, D; Ramírez-Solís, R; Varela-Echavarría, A; Martínez-Rodríguez, H G; Barrera-Saldaña, H A

    1990-01-01

    We have joined the promoter-less sequences of the three hPL genes (hPL-1, hPL-3 and hPL-4) to strong transcriptional control elements. in vivo 35S-labeled proteins from the culture medium of cells transfected with the genes were resolved on SDS-polyacrylamide gels. The presence of characteristic labeled bands, visualized by autoradiography, determined that hPL-4 and hPL-3, but not hPL-1, contribute to the production of mature hPL. In these experiments hPL-3 expressed more RNA and protein than hPL-4. By exchanging the first two exons among hPL and hGH genes, we determined that the abundance of chimeric proteins depended on the genetic origin of the first two exons. Finally, we found evidence indicating that the splice mutation (G----A) at the beginning of the second intron of hPL-1, is not the only cause of the apparent lack of inactivity of this gene, since its reversion does not restore expression. Images PMID:2395633

  18. Liver suction-mediated transfection in mice using a pressure-controlled computer system.

    PubMed

    Shimizu, Kazunori; Zhang, Guangyuan; Kawakami, Shigeru; Taniguchi, Yota; Hayashi, Kouji; Hashida, Mitsuru; Konishi, Satoshi

    2014-01-01

    We previously developed an in vivo tissue suction-mediated transfection method (denoted as the tissue suction method) for naked nucleic acids, such as plasmid DNA (pDNA) and small interfering RNA (siRNA), in mice. However, it remains unclear whether the suction pressure conditions affect the results of this method. Therefore, in the present study, we assembled a computer system to control the suction pressure and investigate the effects of the suction pressure conditions on the efficiency of the liver suction transfection of naked pDNA that encodes luciferase in mice. Using the developed system, we examined the effects of the minimum magnitude of the suction pressure, suction pressure waveform, and suction times of the luciferase expression level in mice livers. We determined that the liver suction method at 5 kPa was not only effective but also caused the lowest hepatic toxicity in mice. Additionally, the results indicated that the suction pressure waveform affects the luciferase expression levels, and a single period of suction on the targeted portion of the liver is sufficient for transfection. Thus, the developed system is useful for performing the tissue suction method with high accuracy and safety.

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

    PubMed

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

    2010-07-01

    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. 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. 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. 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.

  20. 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

  1. Pituitary abscess after autologous bone marrow transplantation.

    PubMed

    Leff, R S; Martino, R L; Pollock, W J; Knight, W A

    1989-05-01

    The first case of pituitary abscess arising in a patient during recovery from autologous bone marrow transplantation is reported. A 31-year-old man with a 9 month history of T-cell lymphoma died suddenly more than 60 days after successful treatment with high-dose cyclophosphamide, total body irradiation, and autologous bone marrow infusion. Autopsy revealed a pituitary abscess associated with clinically silent sphenoid sinusitis. Unique aspects of this case are presented and clinical and pathologic features of pituitary abscess are reviewed. Although rare, pituitary abscess may complicate recovery from bone marrow transplantation.

  2. Autologous fibrin adhesive in experimental tubal anastomosis.

    PubMed

    Rajaram, S; Rusia, U; Agarwal, S; Agarwal, N

    1996-01-01

    To evaluate autologous fibrin in rabbit oviduct anastomosis versus 7-0 vikryl, a conventional suture material used in tubal anastomosis. Thrombin was added to the autologous fibrinogen at the site of anastomosis to obtain a tissue adhesive. The anastomotic time, pregnancy rate, and litter size were evaluated. Three months later, a relaparotomy was done to evaluate patency and degree of adhesions, and a tubal biopsy was taken from the site of anastomosis. Analysis of results showed a statistically significant (P < .001) shortened anastomotic time and superior histopathological union in the tissue adhesive group. Patency rate, pregnancy rate, and degree of adhesions were comparable in both groups.

  3. Inclusion of the helper lipid dioleoyl-phosphatidylethanolamine in solid lipid nanoparticles inhibits their transfection efficiency.

    PubMed

    de Jesus, Marcelo B; Radaic, Allan; Hinrichs, Wouter L J; Ferreira, Carmen V; de Paula, Eneida; Hoekstra, Dick; Zuhorn, Inge S

    2014-02-01

    Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that are suitable carriers for nucleic acids (DNA, siRNA). Considering the beneficial effect of helper lipids on the transfection efficiency with cationic liposomes, the effect of the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on transfection with cationic lipid-containing solid lipid nanoparticles was investigated in PC3 prostate cancer cells. The inclusion of DOPE in SLN formulations, instead of promoted, strongly inhibited SLN transfection efficiency, by frustrating the accommodation of DNA by the particles, as was revealed by biochemical analysis. SLNs devoid of DOPE maintained a homogenous size distribution of approximately 150 nm following lipoplex assembly and cellular delivery, and showed transfection efficiency comparable to that of Lipofectamine 2000' (LF2k). Moreover, the SLNs maintain their high transfection efficiency after lyophilization and long-term storage (1-2 years), an important asset for biomedical applications. There is even the possibility to lyophilize the SLN carrier together with its DNA cargo, which represents an interesting pharmaceutical advantage of the SLN formulations over LF2k. These results reflect marked differences between the physicochemical properties of cationic liposomes and SLNs, the latter requiring more critical lipid-depending properties for effective 'packaging' of DNA but displaying a higher storage stability than cationic lipid based carriers like LF2k.

  4. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring.

    PubMed

    de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, Friedemann; Wirth, Manfred; Wagner, Roland

    2014-05-01

    Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL(-1) 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide

  5. CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

    PubMed Central

    Han, Xin; Liu, Zongbin; Jo, Myeong chan; Zhang, Kai; Li, Ying; Zeng, Zihua; Li, Nan; Zu, Youli; Qin, Lidong

    2015-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system. PMID:26601238

  6. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone

    SciTech Connect

    Adachi, A.; Gendelman, H.E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin, M.A.

    1986-08-01

    The authors considered an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.

  7. Effect of autologous adipose-derived stem cells in renal cold ischemia and reperfusion injury.

    PubMed

    Wang, Y-L; Li, G; Zou, X-F; Chen, X-B; Liu, T; Shen, Z-Y

    2013-11-01

    The aim of this study was to investigate the effect of autologous adipose-derived stem cells (ADSCs) on renal cold ischemia and reperfusion (I/R) injury via intravenous infusion on rats. A renal cold I/R injury rat model was established. Rats were equally randomized into Sham group, Cold I/R group (cold I/R plus culture medium only), and ADSC-treated group (cold I/R plus immediate intrarenal administration of 2 × 10(6) autologous ADSCs, followed by intravenous autologous ADSCs 6 hours after reperfusion). All rats were killed 24 hours after the I/R procedure. Serum creatinine levels were significantly reduced in the ADSC-treated group compared with the Cold I/R group (P < .01). The renal tissue in the ADSC-treated group had well conserved renal architecture compared with the Cold I/R group. The mRNA expression of tumor necrosis factor α was significantly lower and Bcl-2 was higher in the ADSC-treated group than in the Cold I/R group (P < .05). Autologous ADSC infusions ameliorated renal damage undergoing cold I/R injury and improved the renal function, partly through inhibiting inflammatory reactions and reducing apoptosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Enhanced operation of femtosecond lasers and applications in cell transfection.

    PubMed

    Brown, Christian T A; Stevenson, David J; Tsampoula, Xanthi; McDougall, Craig; Lagatsky, Alexander A; Sibbett, Wilson; Gunn-Moore, Frank J; Dholakia, Kishan

    2008-08-01

    In this work we present a review and discussion on the enhancement of femtosecond (fs) lasers for use within biophotonics with a particular focus on their use in optical transfection techniques. We describe the broad range of source options now available for the generation of femtosecond pulses before briefly reviewing the application of fs laser in optical transfection studies. We show that major performance enhancements may be obtained by optimising the spatial and temporal performance of the laser source before considering possible future directions in this field. In relation to optical transfection we describe how such laser sources initiate a multiphoton process to permeate the cell membrane in a transient fashion. We look at aspects of this technique including the ability to combine transfection with optical trapping. For future implementation of such transfection we explore the role of new sources and "nondiffracting" light fields.

  9. Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va

    PubMed Central

    EDGAR, ALASDAIR J.; BENNETT, JONATHAN P.

    1999-01-01

    In mice a molecular motor of the myosin V class (designated myosin Va) is known to be the product of the dilute locus, where a mutation prevents melanosome transport in melanocytes. There is conflicting evidence about whether it has a role in dendrite outgrowth. We investigated its role by transiently transfecting antisense oligonucleotides to inhibit its expression in a melanocyte cell line. We demonstrated mRNA and protein expression of myosin Va in 3 mouse melanocyte lines and 1 human melanoma cell line, using RT-PCR and immunoblotting. Two splice variants were found in human cells whilst only the longer transcript, containing an additional exon, was present in mouse melanocyte lines. The shorter variant was detected in other mouse tissues. Myosin Va protein levels were similar in 3 melanocyte lines with differing amounts of pigmentation, indicating that expression of myosin Va is not tightly coupled to expression of melanin. Immunocytochemistry showed 2 types of myosin Va localisation. A punctate pattern of staining concentrated in the perinuclear region was indicative of organelle association, and the observation of occasional linear punctate staining aligned with F-actin bundles supported the idea that myosin Va has a role in transporting melanosomes along actin filaments. Staining was also intense at tips of dendrites and at sites of dendrite-cell contact, consistent with a possible role in dendrite growth. Transient transfection of antisense phosphorothioate oligodeoxynucleotides targeted against myosin Va mRNA reduced expression of myosin Va protein in cultured mouse melan-a melanocytes by over 70% 20 h after transfection whereas a control (shuffled sequence) oligonucleotide did not. Upon trypsinisation and replating these cells the capacity of the transfected cells to extend new dendrites was reduced in the cells containing the specific antisense oligonucleotides but unaffected by the control oligonucleotide. Image analysis confirmed that the effect of

  10. Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

    PubMed

    Kondo, Shingo; Hongama, Keita; Hanaya, Kengo; Yoshida, Ryota; Kawanobe, Takaaki; Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

    2015-12-15

    Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes

  11. 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.

  12. Cord Blood Banking Standards: Autologous Versus Altruistic.

    PubMed

    Armitage, Sue

    2015-01-01

    Cord blood (CB) is either donated to public CB banks for use by any patient worldwide for whom it is a match or stored in a private bank for potential autologous or family use. It is a unique cell product that has potential for treating life-threatening diseases. The majority of CB products used today are for hematopoietic stem cell transplantation and are accessed from public banks. CB is still evolving as a hematopoietic stem cell source, developing as a source for cellular immunotherapy products, such as natural killer, dendritic, and T-cells, and fast emerging as a non-hematopoietic stem cell source in the field of regenerative medicine. This review explores the regulations, standards, and accreditation schemes that are currently available nationally and internationally for public and private CB banking. Currently, most of private banking is under regulated as compared to public banking. Regulations and standards were initially developed to address the public arena. Early responses from the medical field regarding private CB banking was that at the present time, because of insufficient scientific data to support autologous banking and given the difficulty of making an accurate estimate of the need for autologous transplantation, private storage of CB as "biological insurance" should be discouraged (1, 2, 3). To ensure success and the true realization of the full potential of CB, whether for autologous or allogeneic use, it is essential that each and every product provided for current and future treatments meets high-quality, international standards.

  13. Cord Blood Banking Standards: Autologous Versus Altruistic

    PubMed Central

    Armitage, Sue

    2016-01-01

    Cord blood (CB) is either donated to public CB banks for use by any patient worldwide for whom it is a match or stored in a private bank for potential autologous or family use. It is a unique cell product that has potential for treating life-threatening diseases. The majority of CB products used today are for hematopoietic stem cell transplantation and are accessed from public banks. CB is still evolving as a hematopoietic stem cell source, developing as a source for cellular immunotherapy products, such as natural killer, dendritic, and T-cells, and fast emerging as a non-hematopoietic stem cell source in the field of regenerative medicine. This review explores the regulations, standards, and accreditation schemes that are currently available nationally and internationally for public and private CB banking. Currently, most of private banking is under regulated as compared to public banking. Regulations and standards were initially developed to address the public arena. Early responses from the medical field regarding private CB banking was that at the present time, because of insufficient scientific data to support autologous banking and given the difficulty of making an accurate estimate of the need for autologous transplantation, private storage of CB as “biological insurance” should be discouraged (1, 2, 3). To ensure success and the true realization of the full potential of CB, whether for autologous or allogeneic use, it is essential that each and every product provided for current and future treatments meets high-quality, international standards. PMID:26779485

  14. Autologous Diced Cartilage in Nasal Septoplasty

    PubMed Central

    Sersar, Sameh Ibrahim; Yassin, Ibrahim; Eldin Aly, Mohammed Saad

    2016-01-01

    Diced rib cartilage is an acceptable option in severe nasal deformities. We present our preliminary experience in KAMC in nasal septoplasties using the autologous diced costal cartilage. This is a retrospective study of the 22 cases who needed the autologous diced costal cartilage in our centre in 4 years. All our patients needed autologous diced rib cartilages. Twelve were wrapped with temporalis fascia, eight needed rectus fascia and perichondrium was used in only 2 cases. The naso-frontal angle for the whole series decreased by a mean of 4.41° (p=0.008) for the group using the rectus fascia diced cartilage graft. From the aesthetic point of view, all cases were satisfied except 3 (13.6%); two in the group of diced cartilage temporalis fascia; group 1. From the functional breathing view, only 1 case was not satisfied. He was in group 1. Autologous rib cartilage was shown to be a good graft in nasal septoplasty especially if wrapped with rectus fascia. PMID:27853694

  15. Autologous blood products in rotator cuff repair.

    PubMed

    Mei-Dan, Omer; Carmont, Michael R

    2012-01-01

    We review the management of rotator cuff tears, the mechanism of action of autologous blood products, principally platelet-rich plasma, and the current evidence for effective use of platelet-rich plasma, particularly in relation to the shoulder and chronic rotator cuff tears, for biological augmentation of rotator cuff repair.

  16. Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

    PubMed Central

    Vance, V B; Thompson, E A; Bowman, L H

    1985-01-01

    Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences. Images PMID:2997749

  17. Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

    PubMed

    Boizet, B; Flickinger, J L; Chassy, B M

    1988-12-01

    A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.

  18. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  19. Hydrophobic Moiety of Cationic Lipids Strongly Modulates Their Transfection Activity

    SciTech Connect

    Koynova, Rumiana; Tenchov, Boris; Wang, Li; MacDonald, Robert C.

    2010-01-18

    Synthetic cationic lipids are widely used components of nonviral gene carriers, and the factors regulating their transfection efficiency are the subject of considerable interest. In view of the important role that electrostatic interactions with the polyanionic nucleic acids play in formation of lipoplexes, a common empirical approach to improving transfection has been the synthesis and testing of amphiphiles with new versions of positively charged polar groups, while much less attention has been given to the role of the hydrophobic lipid moieties. On the basis of data for {approx}20 cationic phosphatidylcholine (PC) derivatives, here we demonstrate that hydrocarbon chain variations of these lipids modulate by over 2 orders of magnitude their transfection efficiency. The observed molecular structure-activity relationship manifests in well-expressed dependences of activity on two important molecular characteristics, chain unsaturation and total number of carbon atoms in the lipid chains, which is representative of the lipid hydrophobic volume and hydrophilic-lipophilic ratio. Transfection increases with decrease of chain length and increase of chain unsaturation. Maximum transfection was found for cationic PCs with monounsaturated 14:1 chains. It is of particular importance that the high-transfection lipids strongly promote cubic phase formation in zwitterionic membrane phosphatidylethanolamine (PE). These remarkable correlations point to an alternative, chain-dependent process in transfection, not related to the electrostatic cationic-anionic lipid interactions.

  20. A reverse transfection technology to genetically engineer adult stem cells.

    PubMed

    Okazaki, Arimichi; Jo, Jun-Ichiro; Tabata, Yasuhiko

    2007-02-01

    A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.

  1. [VEGF gene expression in transfected human multipotent stromal cells].

    PubMed

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  2. 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

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

    PubMed

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

    2016-11-01

    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.

  4. Feasibility and effect of ultrasound microbubble-mediated wild-type p53 gene transfection of HeLa cells

    PubMed Central

    CHEN, WEN-JUAN; XIONG, ZHENG-AI; TANG, YAN; DONG, PEI-TING; LI, PAN; WANG, ZHI-GANG

    2012-01-01

    Gene therapy holds great promise for the treatment of diseases. The key problem of gene therapy is the choice of an effective vector. Ultrasound-mediated microbubble technique (UMMT) has already shown promising applications in numerous types of tumors apart from cervical carcinoma. In the present study, according to the results of an MTT assay, we initially chose an ultrasound intensity of 0.5 W/cm2, an ultrasound exposure time of 30 sec and a microbubble concentration of 10% as the optimum experimental condition for wtp53 plasmid transfection into HeLa cells. To further investigate the transfection efficiency of ultrasound combined with microbubbles, RT-PCR analysis was used to examine the mRNA level of p53. The transfection efficiency in the plasmid plus microbubbles and ultrasound group was significantly higher than that of the other groups. Following transfection of the wtp53 gene, flow cytometric analysis showed that the cell cycle of HeLa cells was arrested in the G1 phase. The results of the present study suggest that UMMT, a new gene delivery system, increases the transfection efficiency of the wtp53 gene. Moreover, the growth of HeLa cells was arrested by introducing wtp53. This study may afford a new trend for the gene therapy of cervical carcinoma. PMID:22970006

  5. GDNF-Transfected Macrophages Produce Potent Neuroprotective Effects in Parkinson's Disease Mouse Model

    PubMed Central

    Zhao, Yuling; Haney, Matthew J.; Gupta, Richa; Bohnsack, John P.; He, Zhijian; Kabanov, Alexander V.; Batrakova, Elena V.

    2014-01-01

    The pathobiology of Parkinson's disease (PD) is associated with the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) projecting to the striatum. Currently, there are no treatments that can halt or reverse the course of PD; only palliative therapies, such as replacement strategies for missing neurotransmitters, exist. Thus, the successful brain delivery of neurotrophic factors that promote neuronal survival and reverse the disease progression is crucial. We demonstrated earlier systemically administered autologous macrophages can deliver nanoformulated antioxidant, catalase, to the SNpc providing potent anti-inflammatory effects in PD mouse models. Here we evaluated genetically-modified macrophages for active targeted brain delivery of glial cell-line derived neurotropic factor (GDNF). To capitalize on the beneficial properties afforded by alternatively activated macrophages, transfected with GDNF-encoded pDNA cells were further differentiated toward regenerative M2 phenotype. A systemic administration of GDNF-expressing macrophages significantly ameliorated neurodegeneration and neuroinflammation in PD mice. Behavioral studies confirmed neuroprotective effects of the macrophage-based drug delivery system. One of the suggested mechanisms of therapeutic effects is the release of exosomes containing the expressed neurotropic factor followed by the efficient GDNF transfer to target neurons. Such formulations can serve as a new technology based on cell-mediated active delivery of therapeutic proteins that attenuate and reverse progression of PD, and ultimately provide hope for those patients who are already significantly disabled by the disease. PMID:25229627

  6. Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons.

    PubMed

    Yoon, Seung Yong; Choi, Jung Eun; Hwang, Onyou; Hong, Hea Nam; Lee, Heuiran; Kim, Yoo Kyum; Cho, Sung-Woo; Kim, Hyun; Kim, DongHou

    2004-08-31

    RNA interference is an important tool for gene silencing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against doublecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons.

  7. Reverse transfected cell microarrays in infectious disease research.

    PubMed

    Konrad, Andreas; Jochmann, Ramona; Kuhn, Elisabeth; Naschberger, Elisabeth; Chudasama, Priya; Stürzl, Michael

    2011-01-01

    Several human pathogenic viruses encode large genomes with often more than 100 genes. Viral pathogenicity is determined by carefully orchestrated co-operative activities of several different viral genes which trigger the phenotypic functions of the infected cells. Systematic analyses of these complex interactions require high-throughput transfection technology. Here we have provided a laboratory manual for the reverse transfected cell microarray (RTCM; alternative name: cell chip) as a high-throughput transfection procedure, which has been successfully applied for the systematic analyses of single and combination effects of genes encoded by the human herpesvirus-8 on the NF-kappaB signal transduction pathway. In order to quantitatively determine the effects of viral genes in transfected cells, protocols for the use of GFP as an indicator gene and for indirect immunofluorescence staining of cellular target proteins have been included. RTCM provides a useful methodological approach to investigate systematically combination effects of viral genes on cellular functions.

  8. DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection.

    PubMed

    Clima, Lilia; Peptanariu, Dragos; Pinteala, Mariana; Salic, Adrian; Barboiu, Mihail

    2015-12-25

    Dynamic constitutional frameworks, based on squalene, PEG and PEI components, reversibly connected to core centers, allow the efficient identification of adaptive vectors for good DNA transfection efficiency and are well tolerated by mammalian cells.

  9. 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.

  10. A toolbox facilitating stable transfection of Eimeria species.

    PubMed

    Clark, Julie D; Billington, Karen; Bumstead, Janene M; Oakes, Richard D; Soon, Puay Eng; Sopp, Paul; Tomley, Fiona M; Blake, Damer P

    2008-11-01

    Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.

  11. Histone H2A significantly enhances in vitro DNA transfection.

    PubMed Central

    Balicki, D.; Beutler, E.

    1997-01-01

    BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery. PMID:9407553

  12. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines

    PubMed Central

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K.

    2013-01-01

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [3H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [3H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. PMID:23262422

  13. Circulating microRNAs as Biomarkers for Detection of Autologous Blood Transfusion

    PubMed Central

    Leuenberger, Nicolas; Schumacher, Yorck Olaf; Pradervand, Sylvain; Sander, Thomas; Saugy, Martial; Pottgiesser, Torben

    2013-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs measured in blood plasma have emerged as specific and sensitive markers of physiological processes and disease. In this study, we investigated whether circulating miRNAs can serve as biomarkers for the detection of autologous blood transfusion, a major doping technique that is still undetectable. Plasma miRNA levels were analyzed using high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after autologous blood transfusion (blood bag storage time 42 days) in 10 healthy subjects and 10 controls without transfusion. Other serum markers of erythropoiesis were determined in the same samples. Our results revealed a distinct change in the pattern of circulating miRNAs. Ten miRNAs were upregulated in transfusion samples compared with control samples. Among these, miR-30b, miR-30c, and miR-26b increased significantly and showed a 3.9-, 4.0-, and 3.0-fold change, respectively. The origin of these miRNAs was related to pulmonary and liver tissues. Erythropoietin (EPO) concentration decreased after blood reinfusion. A combination of miRNAs and EPO measurement in a mathematical model enhanced the efficiency of autologous transfusion detection through miRNA analysis. Therefore, our results lay the foundation for the development of miRNAs as novel blood-based biomarkers to detect autologous transfusion. PMID:23840438

  14. Autologous antibodies that bind neuroblastoma cells.

    PubMed

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  15. [Cartilage biopsy for autologous chondrocyte implantation (ACI)].

    PubMed

    Pestka, J M; Salzmann, G M; Südkamp, N P; Niemeyer, P

    2013-06-01

    Autologous chondrocyte implantation (ACI) is an established two-step procedure for the treatment of full-thickness cartilage defects of the knee. Cartilage harvest from the affected knee joint represents the first step of this procedure and is essential for further in vitro expansion of autologous chondrocytes. Nevertheless, the cartilage biopsy process itself is underrepresented in the scientific literature and currently there is only a limited amount of data available addressing this process. Biopsy location as well as the technique itself and instruments used for cartilage collection are not well defined and only little standardisation can be found. The article describes the relevant aspects of the biopsy in the context of ACI with regard to the literature available. Follow-up studies to better define and standardise the cartilage biopsy process are thus required.

  16. Autologous split peroneus longus lateral ankle stabilization.

    PubMed

    Budny, Adam M; Schuberth, John M

    2012-01-01

    Lateral ankle instability is a common clinical entity, and a variety of surgical procedures are available for stabilization after conservative management fails. Herein the authors reviewed outcomes after performing autologous split peroneus longus lateral ankle stabilization, using a previously described surgical technique to anatomically recreate the anterior talofibular and calcaneofibular ligaments. Twenty-five consecutive patients from 2 surgeons' practices underwent reconstruction between March 2007 and January 2011 with a minimum follow-up of 12 (range 12 to 51) months (mean 29.5 months). Follow-up interviews demonstrated 92.0% good or excellent outcomes with only 8.0% rating the outcome as fair and none as poor; 92.0% had no recurrent sprains or difficulty going up or down hills; 88.0% related no difficulty with uneven ground. The authors conclude that the autologous split peroneus longus lateral ankle stabilization results in a stable ankle with a low rate of complications and high patient satisfaction.

  17. [Integrated autologous fat graft in face recontouring].

    PubMed

    Xie, Yun; Zheng, Dan-Ning; Liu, Kai; Gu, Bin; Li, Qing-Feng

    2010-05-01

    To discuss the integrated autologous fat graft technique in face recontouring. In this study we treated 83 cases of face recontouring with 3L3M technique (low pressure suction, low speed centrifugation, low volume, multi-plane, multi-tunnel, multi-point injection). Each case was treated 1-3 times and the interval period is 3-6 months. The result was based on comparing the photos taken from pre-operation and post operation, observing the expression recovery, cysts, local absorption, and patients self evaluation. Long time follow up showed that fat graft can be alive in the recipient site for long time after 1-3 times autologous fat injection. More than 73.5% patients were satisfactory with the curative effect while less than 4.8% patients were unsatisfactory. 3L3M integrated fat graft technique is an effective and safe treatment in face recontouring.

  18. Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells. I. Generation of human thyroid peroxidase-reactive T cells and their T cell receptor repertoire.

    PubMed Central

    Martin, A; Magnusson, R P; Kendler, D L; Concepcion, E; Ben-Nun, A; Davies, T F

    1993-01-01

    To develop a model for endogenous thyroid autoantigen presentation, we transfected EBV-transformed B lymphoblastoid cell lines (EBV-LCL), established from patients with autoimmune thyroid disease and normal controls, with cDNA for the human thyroid autoantigen thyroid peroxidase (hTPO). hTPO-antigen presentation to patient peripheral blood T cells was demonstrated after stimulation in vitro for 7 d with irradiated hTPO-transfected or untransfected autologous EBV-LCL. Anti-hTPO-reactive T cells were subsequently cloned in the presence of irradiated, autologous hTPO-transfected EBV-LCL and IL-2.10 T cell-cloned lines exhibited specific hTPO-induced proliferation (stimulation indices of 2.1-7.9) towards autologous hTPO-transfected EBV-LCL, and were subjected to human T cell receptor (hTCR) V gene analysis, using the PCR for the detection of V alpha and V beta hTcR gene families. The results indicated a preferential use of hTCR V alpha 1 and/or V alpha 3 in 9 of the 10 lines. In contrast, hTCR V beta gene family use was more variable. These data demonstrate a model for the endogenous presentation of human thyroid peroxidase in the absence of other thyroid specific antigens. The high frequency of antigen-specific T cells obtained from PBMC using this technique will facilitate further studies at both the functional and hTCR V gene level. Images PMID:7682574

  19. Pyoderma gangrenosum following autologous breast reconstruction

    PubMed Central

    Tuffaha, Sami H.; Robbins, Sanford H.; Bonawitz, Steven C.

    2017-01-01

    Pyoderma gangrenosum (PG) is an uncommon disorder characterized by the development of painful cutaneous ulceration, commonly precipitated by dermal injury at surgical sites. It is a diagnostic challenge as it manifests as necrotizing wounds which are commonly misdiagnosed as postoperative wound infection or ischemia. We discuss the clinical features and histopathological findings that allow for rapid identification of PG following autologous breast reconstruction and suggest an algorithm to aid diagnosis. PMID:28210559

  20. Pyoderma gangrenosum following autologous breast reconstruction.

    PubMed

    Singh, Prateush; Tuffaha, Sami H; Robbins, Sanford H; Bonawitz, Steven C

    2017-02-01

    Pyoderma gangrenosum (PG) is an uncommon disorder characterized by the development of painful cutaneous ulceration, commonly precipitated by dermal injury at surgical sites. It is a diagnostic challenge as it manifests as necrotizing wounds which are commonly misdiagnosed as postoperative wound infection or ischemia. We discuss the clinical features and histopathological findings that allow for rapid identification of PG following autologous breast reconstruction and suggest an algorithm to aid diagnosis.

  1. Recovery of autologous erythrocytes in transfused patients.

    PubMed

    Wallas, C H; Tanley, P C; Gorrell, L P

    1980-01-01

    A microcapillary method utilizing phthalate esters or an ultracentrifuge method are both capable of separating autologous from homologous erythrocytes in polytransfused patients. The microcapillary technique which is readily adaptable to blood bank laboratories provides a previously unavailable method for defining blood group antigen typings in transfused patients. Such typings are of vital importance in the laboratory evaluation of transfused patients with multiple or weak blood group antibodies.

  2. Cryptococcal meningitis post autologous stem cell transplantation.

    PubMed

    Chaaban, S; Wheat, L J; Assi, M

    2014-06-01

    Disseminated Cryptococcus disease occurs in patients with defective T-cell immunity. Cryptococcal meningitis following autologous stem cell transplant (SCT) has been described previously in only 1 patient, 4 months post SCT and while off antifungal prophylaxis. We present a unique case of Cryptococcus meningitis pre-engraftment after autologous SCT, while the patient was receiving fluconazole prophylaxis. A 41-year-old man with non-Hodgkin's lymphoma underwent autologous SCT. Post-transplant prophylaxis consisted of fluconazole 400 mg daily, levofloxacin 500 mg daily, and acyclovir 800 mg twice daily. On day 9 post transplant, he developed fever and headache. Peripheral white blood cell count (WBC) was 700/μL. Magnetic resonance imaging of the brain showed lesions consistent with meningoencephalitis. Cerebrospinal fluid (CSF) analysis revealed a WBC of 39 with 77% lymphocytes, protein 63, glucose 38, CSF pressure 20.5 cmH2 O, and a positive cryptococcal antigen. CSF culture confirmed Cryptococcus neoformans. The patient was treated with liposomal amphotericin B 5 mg/kg intravenously daily, and flucytosine 37.5 mg/kg orally every 6 h. He was switched to fluconazole 400 mg daily after 3 weeks of amphotericin therapy, with sterilization of the CSF with negative CSFCryptococcus antigen and negative CSF culture. Review of the literature revealed 9 cases of cryptococcal disease in recipients of SCT. Median time of onset was 64 days post transplant. Only 3 meningitis cases were described; 2 of them after allogeneic SCT. Fungal prophylaxis with fluconazole post autologous SCT is recommended at least through engraftment, and for up to 100 days in high-risk patients. A high index of suspicion is needed to diagnose and treat opportunistic infections, especially in the face of immunosuppression and despite adequate prophylaxis. Infection is usually fatal without treatment, thus prompt diagnosis and therapy might be life saving.

  3. Autologous fat graft in postmastectomy pain syndrome.

    PubMed

    Caviggioli, Fabio; Maione, Luca; Forcellini, Davide; Klinger, Francesco; Klinger, Marco

    2011-08-01

    Mastectomy with axillary dissection is still one of the most common procedures in oncologic surgery. Unfortunately, a condition of neuropathic pain, termed postmastectomy pain syndrome, can appear after mastectomy. Although evidence regarding the epidemiology of postmastectomy pain syndrome is well researched, an effective therapy is still unknown. The aim of this study was to assess the clinical effectiveness of lipoaspirate graft in the treatment of postmastectomy pain syndrome. From February of 2006 to August of 2008, a total of 113 patients affected by postmastectomy pain syndrome and severe scar retractions were enrolled for this clinical study. Seventy-two patients were treated with autologous fat grafted in painful scars, and 41 patients did not undergo any further surgical procedure. Pain assessment was performed using a visual analogue scale before and after treatment, with a mean follow-up of 13 months. In addition, antalgic drug intake was recorded in the 34 patients who received a surgical treatment. Results were analyzed using the Wilcoxon rank sum test. A significant decrease in pain according to the visual analogue scale was detected in patients treated with autologous fat graft (3.23-point reduction, p = 0.0005). Twenty-eight of 34 patients stopped their analgesic therapy with a significant follow-up (13 months). Autologous fat grafting is a safe, relatively noninvasive, and rapid surgical procedure. The authors' results suggest its effectiveness for treatment of postmastectomy pain syndrome. Therapeutic, II.

  4. Development of autologous blood cell therapies.

    PubMed

    Kim, Ah Ram; Sankaran, Vijay G

    2016-10-01

    Allogeneic hematopoietic stem cell transplantation and blood cell transfusions are performed commonly in patients with a variety of blood disorders. Unfortunately, these donor-derived cell therapies are constrained due to limited supplies, infectious risk factors, a lack of appropriately matched donors, and the risk of immunologic complications from such products. The use of autologous cell therapies has been proposed to overcome these shortcomings. One can derive such therapies directly from hematopoietic stem and progenitor cells of individuals, which can then be manipulated ex vivo to produce the desired modifications or differentiated to produce a particular target population. Alternatively, pluripotent stem cells, which have a theoretically unlimited self-renewal capacity and an ability to differentiate into any desired cell type, can be used as an autologous starting source for such manipulation and differentiation approaches. Such cell products can also be used as a delivery vehicle for therapeutics. In this review, we highlight recent advances and discuss ongoing challenges for the in vitro generation of autologous hematopoietic cells that can be used for cell therapy. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  5. Autologous platelet-labeling in thrombocytopenia

    SciTech Connect

    Sinzinger, H.; Virgolini, I.; Vinazzer, H. )

    1990-11-01

    Field studies performed with peripheral platelets obtained from 6 male volunteers aged 23 to 29 years revealed an extraordinary dependence of labeling efficiency on incubation time and platelet concentration after {sup 111}In-oxine platelet labeling. Since the monitoring of in vivo-platelet function in patients with thrombocytopenia may cause problems due to insufficient labeling results and homologous platelets may show a different in vivo behaviour to autologous ones, we have searched for the minimal amount of platelets necessary to allow appropriate labeling and imaging in patients with thrombocytopenia. In 15 patients with untreated thrombocytopenia aged 14 to 79 years demonstrating a mean peripheral platelet count of 2.509 +/- 1.45 x 10(4) cells/microliters autologous {sup 111}In-oxine platelet labeling was performed. The results indicate that approximately 1 x 10(8) (concentrated) platelets/ml are necessary to obtain an adequate labeling efficiency and recovery. This platelet concentration can be easily achieved by drawing one more Monovette of whole blood per each 5 x 10(4) platelets/microliter peripheral platelet count less than 2 x 10(5)/microliter. It is concluded, that calculation of the required number of platelets in advance, variation of the blood volume drawn and the volume of incubation buffer allow informative, qualitative and quantitative results using autologous platelets. The method presented effectively circumvents the requirement of homologous platelets for radiolabeling in thrombocytopenia.

  6. RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.

    PubMed

    Böttger, Jan; Arnold, Katrin; Thiel, Carlo; Rennert, Christiane; Aleithe, Susanne; Hofmann, Ute; Vlaic, Sebastian; Sales, Susanne; Shevchenko, Andrej; Matz-Soja, Madlen

    2015-09-01

    Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4%), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

  7. Dystrophic calcifications after autologous fat injection on face.

    PubMed

    Kim, Dai Hyun; Jang, Hee Won; Kim, Hee Joo; Son, Sang Wook

    2014-06-01

    Autologous fat injection is widely used procedure for various functional and aesthetic purposes. However, it could result in many immediate or delayed complications including dystrophic calcifications. Almost all of the case reports about dystrophic calcification after autologous fat injection were result from the iatrogenic tissue trauma of breast augmentation. This is a report of a 30-year-old patient who developed pathologically proven multiple dystrophic calcifications on the face after autologous fat injection.

  8. Development of an Autologous Macrophage-based Adoptive Gene Transfer Strategy to Treat Posttraumatic Osteoarthritis (PTOA) and Osteoarithritis (OA)

    DTIC Science & Technology

    2015-02-01

    macrophage-based adoptive gene therapy with both an anti-catabolic gene ( IL -1ra or IL -1β shRNA) and a pro-chondrogenic gene (TGFβ3) is based on the...deliver and confine expression of an anti-catabolic gene ( IL -1ra or IL -1β shRNA) along with a chondrogenic gene (TGFβ3) in the inflamed areas within the...synovium of the PTOA joint; and 2) the IL -1ra (or IL -1β shRNA) and TGFβ3 combination autologous macrophage-based adoptive gene transfer strategy will

  9. Autologous Chondrocytes and Next-Generation Matrix-Based Autologous Chondrocyte Implantation.

    PubMed

    Hinckel, Betina B; Gomoll, Andreas H

    2017-07-01

    Focal chondral defects of the knee are common and can significantly impair quality of life. The autologous chondrocyte implantation technique has evolved over the past 20 years; the newest third-generation technique is matrix-induced autologous chondrocyte implantation. Physical examination is important to characterize location and source of pain and identify associated injuries. Imaging studies allow characterization of the lesions, identification of associated lesions, and alignment. Conservative measures should be exhausted before proceeding with surgical treatment. Steps of surgical treatment are diagnostic arthroscopy and biopsy, chondrocyte culture, and chondrocyte implantation. The techniques and their outcomes are discussed in this article. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Autologous Growth Factor Injections in Chronic Tendinopathy

    PubMed Central

    Sandrey, Michelle A.

    2014-01-01

    Reference: de Vos RJ, van Veldhoven PLJ, Moen MH, Weir A, Tol JL. Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull. 2010;95:63–77. Clinical Question: The authors of this systematic review evaluated the literature to critically consider the effects of growth factors delivered through autologous whole-blood and platelet-rich–plasma (PRP) injections in managing wrist-flexor and -extensor tendinopathies, plantar fasciopathy, and patellar tendinopathy. The primary question was, according to the published literature, is there sufficient evidence to support the use of growth factors delivered through autologous whole-blood and PRP injections for chronic tendinopathy? Data Sources: The authors performed a comprehensive, systematic literature search in October 2009 using PubMed, MEDLINE, EMBASE, CINAHL, and the Cochrane library without time limits. The following key words were used in different combinations: tendinopathy, tendinosis, tendinitis, tendons, tennis elbow, plantar fasciitis, platelet rich plasma, platelet transfusion, and autologous blood or injection. The search was limited to human studies in English. All bibliographies from the initial literature search were also viewed to identify additional relevant studies. Study Selection: Studies were eligible based on the following criteria: (1) Articles were suitable (inclusion criteria) if the participants had been clinically diagnosed as having chronic tendinopathy; (2) the design had to be a prospective clinical study, randomized controlled trial, nonrandomized clinical trial, or prospective case series; (3) a well-described intervention in the form of a growth factor injection with either PRP or autologous whole blood was used; and (4) the outcome was reported in terms of pain or function (or both). Data Extraction: All titles and abstracts were assessed by 2 researchers, and all relevant articles were obtained. Two researchers independently read the full text of

  11. Autologous growth factor injections in chronic tendinopathy.

    PubMed

    Sandrey, Michelle A

    2014-01-01

    de Vos RJ, van Veldhoven PLJ, Moen MH, Weir A, Tol JL. Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull. 2010;95:63-77. The authors of this systematic review evaluated the literature to critically consider the effects of growth factors delivered through autologous whole-blood and platelet-rich-plasma (PRP) injections in managing wrist-flexor and -extensor tendinopathies, plantar fasciopathy, and patellar tendinopathy. The primary question was, according to the published literature, is there sufficient evidence to support the use of growth factors delivered through autologous whole-blood and PRP injections for chronic tendinopathy? The authors performed a comprehensive, systematic literature search in October 2009 using PubMed, MEDLINE, EMBASE, CINAHL, and the Cochrane library without time limits. The following key words were used in different combinations: tendinopathy, tendinosis, tendinitis, tendons, tennis elbow, plantar fasciitis, platelet rich plasma, platelet transfusion, and autologous blood or injection. The search was limited to human studies in English. All bibliographies from the initial literature search were also viewed to identify additional relevant studies. Studies were eligible based on the following criteria: (1) Articles were suitable (inclusion criteria) if the participants had been clinically diagnosed as having chronic tendinopathy; (2) the design had to be a prospective clinical study, randomized controlled trial, nonrandomized clinical trial, or prospective case series; (3) a well-described intervention in the form of a growth factor injection with either PRP or autologous whole blood was used; and (4) the outcome was reported in terms of pain or function (or both). All titles and abstracts were assessed by 2 researchers, and all relevant articles were obtained. Two researchers independently read the full text of each article to determine if it met the inclusion criteria. If opinions differed on

  12. Femtosecond cellular transfection using a non-diffracting beam

    NASA Astrophysics Data System (ADS)

    Tsampoula, X.; Garcés-Chávez, V.; Comrie, M.; Stevenson, D. J.; Agate, B.; Brown, C. T. A.; Gunn-Moore, F.; Dholakia, K.

    2008-02-01

    Efficient DNA delivery into single living cells would be a very powerful capability for cell biologists for elucidating basic cellular functions but also in other fields such as applied drug discovery and gene therapy. The ability to gently permeate the cell membrane and introduce foreign DNA with the assistance of lasers is a powerful methodology but requires exact focusing due to the required two-photon power density. Here, we demonstrate a laser-mediated delivery method of the red fluorescent protein DS-RED into Chinese hamster Ovary (CHO) cells. We used an elongated beam of light created by a Bessel beam (BB) which obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a threshold for transfection of 20%, the BB gives us transfection over twenty times the axial distance compared to the Gaussian beam of equivalent core diameter. In addition, by exploiting the BB property of reconstruction, we demonstrate successful transfection of CHO cells which involves the BB passing through an obstructive layer and re forming itself prior to reaching the cell membrane. In the light of this exciting result, one can envisage the possibility of achieving transfection through multiple cell monolayer planes and tissues using this novel light field, eliminating this way the stringent requirements for tight focusing.

  13. Polyarginine Molecular Weight Determines Transfection Efficiency of Calcium Condensed Complexes

    PubMed Central

    Alhakamy, Nabil A.; Berkland, Cory J.

    2014-01-01

    Cell penetrating peptides (CPPs) have been extensively studied in polyelectrolyte complexes as a means to enhance the transfection efficiency of plasmid DNA (pDNA). Increasing the molecular weight of CPPs often enhances gene expression, but poses a risk of increased cytotoxicity and immunogenicity compared to low molecular weight CCPs. Conversely, low molecular weight CPPs typically have low transfection efficiency due to large complex size. Complexes made using low molecular weight CPPs were found to be condensed to a small size by adding calcium. In this study, complexes of low molecular weight polyarginine and pDNA were condensed with calcium. These complexes showed high transfection efficiency and low cytotoxicity in A549 carcinomic human alveolar basal epithelial cells. The relationship between transfection efficiency and polyarginine size (5, 7, 9 or 11 amino acids), polyarginine/pDNA charge ratios, and calcium concentrations were studied. Polyarginine 7 was significantly more effective than other polyarginines under most formulation conditions suggesting a link between cell penetration ability and transfection efficiency. PMID:23534410

  14. 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.

  15. 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.

  16. 96-well electroporation method for transfection of mammalian central neurons.

    PubMed

    Buchser, William J; Pardinas, Jose R; Shi, Yan; Bixby, John L; Lemmon, Vance P

    2006-11-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.

  17. 96-Well electroporation method for transfection of mammalian central neurons

    PubMed Central

    Buchser, William J.; Pardinas, Jose R.; Shi, Yan; Bixby, John L.; Lemmon, Vance P.

    2008-01-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes. PMID:17140120

  18. Synthesis of linear polyethylenimine derivatives for DNA transfection.

    PubMed

    Brissault, Blandine; Kichler, Antoine; Guis, Christine; Leborgne, Christian; Danos, Olivier; Cheradame, Hervé

    2003-01-01

    A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

  19. In trans promoter activation by enhancers in transient transfection.

    PubMed

    Smirnov, N A; Akopov, S B; Didych, D A; Nikolaev, L G

    2017-03-01

    Earlier, it was reported that the strong cytomegalovirus enhancer can activate the cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular process.

  20. Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles.

    PubMed

    Fayazpour, Farzaneh; Lucas, Bart; Alvarez-Lorenzo, Carmen; Sanders, Niek N; Demeester, Jo; De Smedt, Stefaan C

    2006-10-01

    In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.

  1. Fluorescence microscopy-based RNA interference screening.

    PubMed

    Gunkel, Manuel; Beil, Nina; Beneke, Jürgen; Reymann, Jürgen; Erfle, Holger

    2015-01-01

    Using RNAi interference (RNAi), it is possible to study the effect of specific gene knockdowns in mammalian cells. In this protocol we present the automated preparation of "ready to transfect" multiwell plates and cell arrays, on which cells can be grown which are then reversely transfected with one type of siRNA in every individual well or spot. Additionally, different microscope types for screening approaches are compared and considerations about the information workflow are made.

  2. Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

    PubMed

    Sennikov, Sergey Vital'evich; Shevchenko, Julia Alexandrovna; Kurilin, Vasilii Vasil'evich; Khantakova, Julia Nikolaevna; Lopatnikova, Julia Anatol'evna; Gavrilova, Elena Vasil'evna; Maksyutov, Rinat Amirovich; Bakulina, Anastasiya Yur'evna; Sidorov, Sergey Vasil'evich; Khristin, Alexander Alexandrovich; Maksyutov, Amir Zakievich

    2016-02-01

    Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

  3. Autologous stem cell transplantation for systemic sclerosis.

    PubMed

    Farge, Dominique; Nash, Richard; Laar, Jacob M

    2008-12-01

    Systemic sclerosis (SSc) is a generalised autoimmune disease, of yet unknown origin, with two major clinical subsets: the limited (lcSSc) and the diffuse cutaneous (dcSSc) forms, which can be distinguished by the extent of skin involvement, the autoantibody profile and the pattern of organ involvement. With an incidence of 1/10(5), SSc affects around 250,000 people in Europe and is responsible for significant morbidity with a 5-year mortality rate of at least 30% of all patients. In patients with rapidly progressive dcSSc, the 5-year mortality is estimated to be 40-50%. Hematopoietic stem cell transplantation (HSCT), mostly autologous but also allogeneic in some specific cases, has been employed worldwide since 1996 as a new therapeutic strategy in patients with a poor prognosis. In 2007, 150 HSCT procedures have been reported in the EBMT data base. We review herein both the short and the long-term reports from the various European and North American phase I-II studies, which have shown that autologous HSCT in selected patients with severe dcSSc results in sustained improvement of skin thickening and stabilisation of organ function up to seven years after transplantation. Based on these promising results, ongoing phase III trials have been designed in parallel, both in Europe (ASTIS) and in North America (SCOTT) aiming to analyse the respective benefits from autologous HSCT respectively without or with high dose irradiation. This review reports the current data concerning the effects of HSCT on survival, skin, and major organ function in patients with severe dcSSc.

  4. Microinjection and Fluorescence In Situ Hybridization Assay for Studying mRNA Export in Mammalian Cells.

    PubMed

    Wang, Ke; Shi, Min; Cheng, Hong

    2017-01-01

    Microinjection and Fluorescence in situ Hybridization (FISH) assay is a useful method for mRNA export studies, which can overcome the problems of traditional transfection in cells. Here, we describe the method of microinjection and FISH assay applied in investigation of mRNA export. By this method we can estimate the mRNA export kinetics, examining mRNA export in cells with low transfection efficiencies, and observing nuclear export of aberrant RNAs.

  5. Autologous bone marrow transplantation by photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Gulliya, Kirpal S.

    1992-06-01

    Simultaneous exposure of Merocyanine 540 dye containing cultured tumor cells to 514-nm laser light (93.6 J/cm2) results in virtually complete cell destruction. Under identical conditions, 40% of the normal progenitor (CFU-GM) cells survive the treatment. Laser- photoradiation treated, cultured breast cancer cells also were killed, and living tumor cells could not be detected by clonogenic assays or by anti-cytokeratin monoclonal antibody method. Thus, laser photoradiation therapy could be useful for purging of contaminating tumor cells from autologous bone marrow.

  6. Biological effects of eukaryotic recombinant plasmid pReceiver-M61-BAI-1 transfection on T24 cells and HUVECs.

    PubMed

    Tian, Da-Wei; Hu, Hai-Long; Sun, Yan; Tang, Yang; Lei, Ming-De; Liu, Li-Wei; Han, Rui-Fa; Wu, Chang-Li

    2016-08-01

    The aim of the current study was to investigate the biological effect on T24 cells and human umbilical vein endothelial cells (HUVECs) of transfection with brain-specific angiogenesis inhibitor-1 (BAI-1). The recombinant plasmid pReceiver-M61-BAI-1 was transfected into human superficial bladder tumor cells (T24) and HUVECs, in parallel with the vector control. mRNA and protein expression levels of BAI‑1 were then detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Cell apoptosis of T24 cells and HUVECs prior and subsequent to transfection with BAI‑1 was analyzed by flow cytometric analysis. Proliferation of T24 cells and HUVECs prior and subsequent to transfection of BAI-1 was assessed by the MTT method. T24 cells and HUVECs transfected with pReceiver‑M61‑BA1‑1 were classed as the experimental group; T24 cells and HUVECs transfected with p‑Receiver‑M61 were the control group. qPCR and western blotting methods confirmed that there was positive expression of BAI‑1 in T24 cells and HUVECs transfected with pReceiver‑M61‑BAI‑1, however BAI‑1 was not expressed in T24 cells and HUVECs transfected with pReceiver‑M61. The results of the MTT assay demonstrated that absorbance was markedly reduced in HUVECs at 12, 48 and 72 h subsequent to transfection with pReceiver-M61-BAI-1 when compared with that of the control group and in T24 cells transfected with p‑Receiver-M61-BAI-1. Furthermore, flow cytometry results also indicated that the apoptotic rate of HUVECs transfected with p‑Receiver‑M61‑BAI‑1 was significantly increased compared with that of the control group and T24 cells transfected with p‑Receiver‑M61‑BAI‑1. BAI‑1 was observed to markedly inhibit the proliferation of vascular endothelial cells in vitro, however, no direct inhibition by BAI‑1 was observed in T24 cells. In conclusion, BAI-1 is suggested to be a potential novel therapautic target for the inhibition of

  7. Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

    PubMed

    Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

    2017-03-01

    Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

  8. In vivo Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

    PubMed Central

    Shimizu, Kazunori; Kawakami, Shigeru; Hayashi, Kouji; Kinoshita, Hideyuki; Kuwahara, Koichiro; Nakao, Kazuwa; Hashida, Mitsuru; Konishi, Satoshi

    2012-01-01

    We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids. PMID:22844458

  9. Detection of accessory spleens with indium 111-labeled autologous platelets

    SciTech Connect

    Davis, H.H., II; Varki, A.; Heaton, W.A.; Siegel, B.A.

    1980-01-01

    In two patients with recurrent immune thrombocytopenia, accessory splenic tissue was demonstrated by radionuclide imaging following administration of indium 111-labeled autologous platelets. In one of these patients, no accessory splenic tissue was seen on images obtained with technetium 99m sulfur colloid. This new technique provides a simple means for demonstrating accessory spleens and simultaneously evaluating the life-span of autologous platelets.

  10. Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

    PubMed

    Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

    2012-01-01

    Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. Copyright © 2012 S. Karger AG, Basel.

  11. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  12. Transfection of E. coli with lambda DNA by electroporation.

    PubMed

    Magistrelli, C; Colombo, E; Tognoni, A; Grandi, G

    1992-10-01

    In the ambit of the B. subtilis genoma sequencing and mapping project, we have set up an electroporation method to transfect E. coli cells with lambda DNA. This methodology presents features that make it preferable to traditional in vitro packaging for some purposes. Here we will illustrate the experimental procedure and the possible applications.

  13. Detection methods for autologous blood doping.

    PubMed

    Segura, J; Monfort, N; Ventura, R

    2012-11-01

    The use of blood doping is forbidden by the World Anti-Doping Agency. Several practices, such as blood transfusions are used to increase oxygen delivery to muscles and all of them are highly pursued. In this regard, the development of accurate methodologies for detecting these prohibited practices is one of the current aims of the anti-doping control laboratories. Flow cytometry methods are able to detect allogeneic blood transfusions but there is no official methodology available to detect autologous blood transfusions. This paper reviews protocols, including the Athlete Biological Passport, that use indirect markers to detect misuse of blood transfusions, especially autologous blood transfusions. The methods of total haemoglobin mass measurements and the detection of metabolites of blood bags plasticizers in urine are reviewed. The latter seems to be an important step forward because it is a fast screening method and it is based on urine, a fluid widely available for doping control. Other innovative approaches to blood transfusion detection are also mentioned. A combination of the reported methodologies and the implementation of the Athlete Biological Passport is becoming a promising approach.

  14. Hyaline cartilage degenerates after autologous osteochondral transplantation.

    PubMed

    Tibesku, C O; Szuwart, T; Kleffner, T O; Schlegel, P M; Jahn, U R; Van Aken, H; Fuchs, S

    2004-11-01

    Autologous osteochondral grafting is a well-established clinical procedure to treat focal cartilage defects in patients, although basic research on this topic remains sparse. The aim of the current study was to evaluate (1) histological changes of transplanted hyaline cartilage of osteochondral grafts and (2) the tissue that connects the transplanted cartilage with the adjacent cartilage in a sheep model. Both knee joints of four sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the contralateral femoral condyle. The animals were sacrificed after three months and the received knee joints were evaluated histologically. Histological evaluation showed a complete ingrowth of the osseous part of the osteochondral grafts. A healing or ingrowth at the level of the cartilage could not be observed. Histological evaluation of the transplanted grafts according to Mankin revealed significantly more and more severe signs of degeneration than the adjacent cartilage, such as cloning of chondrocytes and irregularities of the articular surface. We found no connecting tissue between the transplanted and the adjacent cartilage and histological signs of degeneration of the transplanted hyaline cartilage. In the light of these findings, long-term results of autologous osteochondral grafts in human beings have to be followed critically.

  15. One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

    SciTech Connect

    Protic-Sabljic, M.; Kraemer, K.H.

    1985-10-01

    The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.

  16. Structure-activity relationship in cationic lipid mediated gene transfection.

    PubMed

    Niculescu-Duvaz, Dan; Heyes, James; Springer, Caroline J

    2003-07-01

    Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.

  17. Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors.

    PubMed Central

    Tobias, E S; Rozengurt, E; Connell, J M; Houslay, M D

    1997-01-01

    Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase. PMID:9291130

  18. Increasing the Dose of Autologous Chondrocytes Improves Articular Cartilage Repair

    PubMed Central

    Guillén-García, Pedro; Rodríguez-Iñigo, Elena; Guillén-Vicente, Isabel; Caballero-Santos, Rosa; Guillén-Vicente, Marta; Abelow, Stephen; Giménez-Gallego, Guillermo

    2014-01-01

    Background: We hypothesized that implanting cells in a chondral defect at a density more similar to that of the intact cartilage could induce them to synthesize matrix with the features more similar to that of the uninjured one. Methods: We compared the implantation of different doses of chondrocytes: 1 million (n = 5), 5 million (n = 5), or 5 million mesenchymal cells (n = 5) in the femoral condyle of 15 sheep. Tissue generated by microfracture at the trochlea, and normal cartilage from a nearby region, processed as the tissues resulting from the implantation, were used as references. Histological and molecular (expression of type I and II collagens and aggrecan) studies were performed. Results: The features of the cartilage generated by implantation of mesenchymal cells and elicited by microfractures were similar and typical of a poor repair of the articular cartilage (presence of fibrocartilage, high expression of type I collagen and a low mRNA levels of type II collagen and aggrecan). Nevertheless, in the samples obtained from tissues generated by implantation of chondrocytes, hyaline-like cartilage, cell organization, low expression rates of type I collagen and high levels of mRNA corresponding to type II collagen and aggrecan were observed. These histological features, show less variability and are more similar to those of the normal cartilage used as control in the case of 5 million cells implantation than when 1 million cells were used. Conclusions: The implantation of autologous chondrocytes in type I/III collagen membranes at high density could be a promising tool to repair articular cartilage. PMID:26069691

  19. Autologous versus allogeneic transfusion: patients' perceptions and experiences

    PubMed Central

    Graham, I D; Fergusson, D; Dokainish, H; Biggs, J; McAuley, L; Laupacis, A

    1999-01-01

    BACKGROUND: Preoperative autologous donation is one way to decrease a patient's exposure to allogeneic blood transfusion. This study was designed to determine patients' perceptions about the autologous blood donation process and their experiences with transfusion. METHODS: To assess patient perception, a questionnaire was administered a few days before surgery to patients undergoing elective cardiac and orthopedic surgery in a Canadian teaching hospital. All patients attending the preoperative autologous donation clinic during a 10-month period were eligible. A convenience sample of patients undergoing the same types of surgery who had not predonated blood were selected from preadmission clinics. Patient charts were reviewed retrospectively to assess actual transfusion practice in all cases. RESULTS: A total of 80 patients underwent cardiac surgery (40 autologous donors, 40 nondonors) and 73 underwent orthopedic surgery (38 autologous donors, 35 nondonors). Of the autologous donors, 75 (96%) attended all scheduled donation appointments, 73 (93%) said that they were "very likely" or "likely" to predonate again, and 75 (96%) said that they would recommend autologous donation to others. There was little difference in preoperative symptoms between the autologous donors and the nondonors, although the former were more likely than the latter to report that their overall health had remained the same during the month before surgery (30 [75%] v. 21 [52%] for the cardiac surgery patients and 30 [79%] v. 18 [51%] for the orthopedic surgery patients). When the autologous donors were asked what they felt their chances would have been of receiving at least one allogeneic blood transfusion had they not predonated, the median response was 80%. When they were asked what their chances were after predonating their own blood, the median response was 0%. The autologous donors were significantly less likely to receive allogeneic blood transfusions (6 [15%] for cardiac surgery and 3 [8

  20. Luminescent-activated transfected killer cells to monitor leukocyte trafficking during systemic bacterial and fungal infection.

    PubMed

    Lin, Lin; Ibrahim, Ashraf S; Baquir, Beverlie; Palosaari, Andrew; Spellberg, Brad

    2012-01-15

    Activated transfected killer (ATAK) cells are immortal phagocytes transfected with a luminescence reporter that effectively treat lethal infections in neutropenic mice. Their in vivo trafficking, lifespan, and immunogenicity are unknown. Mice were made neutropenic; infected or not with Staphylococcus aureus, Acinetobacter baumannii, Candida albicans, or Aspergillus fumigatus; and treated intraperitoneally with ATAK cells. Cell trafficking and lifespan were assessed by in vivo imaging and reverse transcription-polymerase chain reaction. In uninfected neutropenic mice, ATAK cells spread from the mesentery into visceral organs on days 1-3. Splenic accumulation of ATAK cells increased at day 1 after infection with S. aureus and A. baumannii, and kidney accumulation increased in mice infected with C. albicans. Lung accumulation was seen at day 3 in mice infected by inhalation with A. fumigatus. By day 8, coincident with increasing anti-ATAK antibodies, luminescence signal was lost and there was no detectable mRNA transcription from ATAK cells. ATAK cells accumulated in target organs with distinct profiles, depending on the microbial etiology of infection. Finally, generation of an anti-ATAK immune response may provide an important safety mechanism that helps clear the cells from the host as the marrow recovers.

  1. Luminescent-Activated Transfected Killer Cells to Monitor Leukocyte Trafficking During Systemic Bacterial and Fungal Infection

    PubMed Central

    Lin, Lin; Ibrahim, Ashraf S.; Baquir, Beverlie; Palosaari, Andrew

    2012-01-01

    Background. Activated transfected killer (ATAK) cells are immortal phagocytes transfected with a luminescence reporter that effectively treat lethal infections in neutropenic mice. Their in vivo trafficking, lifespan, and immunogenicity are unknown. Methods. Mice were made neutropenic; infected or not with Staphylococcus aureus, Acinetobacter baumannii, Candida albicans, or Aspergillus fumigatus; and treated intraperitoneally with ATAK cells. Cell trafficking and lifespan were assessed by in vivo imaging and reverse transcription–polymerase chain reaction. Results. In uninfected neutropenic mice, ATAK cells spread from the mesentery into visceral organs on days 1–3. Splenic accumulation of ATAK cells increased at day 1 after infection with S. aureus and A. baumannii, and kidney accumulation increased in mice infected with C. albicans. Lung accumulation was seen at day 3 in mice infected by inhalation with A. fumigatus. By day 8, coincident with increasing anti-ATAK antibodies, luminescence signal was lost and there was no detectable mRNA transcription from ATAK cells. Conclusions. ATAK cells accumulated in target organs with distinct profiles, depending on the microbial etiology of infection. Finally, generation of an anti-ATAK immune response may provide an important safety mechanism that helps clear the cells from the host as the marrow recovers. PMID:22124127

  2. Specific Transfection of Inflamed Brain by Macrophages: A New Therapeutic Strategy for Neurodegenerative Diseases

    PubMed Central

    Haney, Matthew J.; Zhao, Yuling; Harrison, Emily B.; Mahajan, Vivek; Ahmed, Shaheen; He, Zhijian; Suresh, Poornima; Hingtgen, Shawn D.; Klyachko, Natalia L.; Mosley, R. Lee; Gendelman, Howard E.; Kabanov, Alexander V.; Batrakova, Elena V.

    2013-01-01

    The ability to precisely upregulate genes in inflamed brain holds great therapeutic promise. Here we report a novel class of vectors, genetically modified macrophages that carry reporter and therapeutic genes to neural cells. Systemic administration of macrophages transfected ex vivo with a plasmid DNA (pDNA) encoding a potent antioxidant enzyme, catalase, produced month-long expression levels of catalase in the brain resulting in three-fold reductions in inflammation and complete neuroprotection in mouse models of Parkinson's disease (PD). This resulted in significant improvements in motor functions in PD mice. Mechanistic studies revealed that transfected macrophages secreted extracellular vesicles, exosomes, packed with catalase genetic material, pDNA and mRNA, active catalase, and NF-κb, a transcription factor involved in the encoded gene expression. Exosomes efficiently transfer their contents to contiguous neurons resulting in de novo protein synthesis in target cells. Thus, genetically modified macrophages serve as a highly efficient system for reproduction, packaging, and targeted gene and drug delivery to treat inflammatory and neurodegenerative disorders. PMID:23620794

  3. Transfection of Antisense Oligonucleotides Mediated by Cationic Vesicles Based on Non-Ionic Surfactant and Polycations Bearing Quaternary Ammonium Moieties

    PubMed Central

    Mayr, Judith; Grijalvo, Santiago; Bachl, Jürgen; Pons, Ramon; Eritja, Ramon; Díaz Díaz, David

    2017-01-01

    Three different ionene polymers with varying quaternary ammonium moieties were used as a proof of concept for the formulation of antisense oligonucleotides, which are capable of inhibiting Renilla luciferase messenger ribonucleic acid (mRNA). Cationic vesicles, consisting of cationic polymer, antisense oligonucleotide (Luc) and non-ionic surfactant polysorbate 80, were investigated regarding their ζ potential, cytotoxicity and transfection efficiency. Deoxyribonucleic acid- (DNA) forming complexes in the presence of cationic vesicles were also investigated in terms of small-angle X-ray scattering (SAXS). The studied cationic vesicles showed very little, if any, toxicity against HeLa cells. Transfection abilities proved to vary strongly depending on the present quaternary ammonium moiety. PMID:28587106

  4. Transfection of Antisense Oligonucleotides Mediated by Cationic Vesicles Based on Non-Ionic Surfactant and Polycations Bearing Quaternary Ammonium Moieties.

    PubMed

    Mayr, Judith; Grijalvo, Santiago; Bachl, Jürgen; Pons, Ramon; Eritja, Ramon; Díaz Díaz, David

    2017-05-26

    Three different ionene polymers with varying quaternary ammonium moieties were used as a proof of concept for the formulation of antisense oligonucleotides, which are capable of inhibiting Renilla luciferase messenger ribonucleic acid (mRNA). Cationic vesicles, consisting of cationic polymer, antisense oligonucleotide (Luc) and non-ionic surfactant polysorbate 80, were investigated regarding their ζ potential, cytotoxicity and transfection efficiency. Deoxyribonucleic acid- (DNA) forming complexes in the presence of cationic vesicles were also investigated in terms of small-angle X-ray scattering (SAXS). The studied cationic vesicles showed very little, if any, toxicity against HeLa cells. Transfection abilities proved to vary strongly depending on the present quaternary ammonium moiety.

  5. In vitro transfection mediated by dendrigraft poly(L-lysines): the effect of structure and molecule size.

    PubMed

    Hofman, Jakub; Buncek, Martin; Haluza, Radovan; Streinz, Ludvik; Ledvina, Miroslav; Cigler, Petr

    2013-02-01

    Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Rapid desensitization with autologous sweat in cholinergic urticaria.

    PubMed

    Kozaru, Takeshi; Fukunaga, Atsushi; Taguchi, Kumiko; Ogura, Kanako; Nagano, Tohru; Oka, Masahiro; Horikawa, Tatsuya; Nishigori, Chikako

    2011-09-01

    The majority of patients with cholinergic urticaria presents with strong hypersensitivity to autologous sweat. Patients with severe cholinergic urticaria are frequently resistant to H(1) antagonists which are used in conventional therapies for various types of urticaria. It has been reported that desensitization using partially purified sweat antigen was effective in a patient with cholinergic urticaria. The aim of this study is to determine the usefulness of rapid desensitization with autologous sweat in severe cholinergic urticaria, because rapid desensitization has proven to be a quick and effective immunotherapy for allergies to various allergens. Six patients with severe cholinergic urticaria who are resistant to H(1) antagonists and have sweat hypersensitivity were enrolled in a rapid desensitization protocol. In all six patients, the responses for skin tests with autologous sweat were attenuated after rapid desensitization with autologous sweat. Two of the three cholinergic urticaria patients showed reduced histamine release with autologous sweat after the rapid desensitization with autologous sweat. Further, the rapid desensitization and subsequent maintenance treatment reduced the symptoms in five of the six patients. This study provides evidence that rapid desensitization with autologous sweat is beneficial for treating cholinergic urticaria patients resistant to conventional therapy who have sweat hypersensitivity.

  7. Autologous Chondrocyte Implantation: Past, Present, and Future.

    PubMed

    Welch, Tyler; Mandelbaum, Bert; Tom, Minas

    2016-06-01

    Focal cartilage defects of the knee are relatively common and may increase the risk of developing osteoarthritis. Autologous chondrocyte implantation (ACI) aims to restore the integrity of isolated cartilage lesions through the induction of hyaline-like cartilage formation. Although ACI has traditionally been used as a second-line treatment, recent evidence suggests that ACI should be considered as a first-line treatment option in certain patients. Recent controlled trials also suggest that there are improved clinical outcomes among those patients who undergo ACI over the mid-term and long-term compared with those treated with microfracture or osteochondral autograft/mosaicplasty, regardless of lesion size. Recent literature also indicates that arthroscopic, second-generation and third-generation techniques are associated with better outcomes and fewer complications than first-generation ACI. In summary, ACI is an effective tool for cartilage restoration that may be more efficacious and durable than other cartilage restoration techniques for appropriate candidates.

  8. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV

    PubMed Central

    Tebas, Pablo; Stein, David; Tang, Winson W.; Frank, Ian; Wang, Shelley Q.; Lee, Gary; Spratt, S. Kaye; Surosky, Richard T.; Giedlin, Martin A.; Nichol, Geoff; Holmes, Michael C.; Gregory, Philip D.; Ando, Dale G.; Kalos, Michael; Collman, Ronald G.; Binder-Scholl, Gwendolyn; Plesa, Gabriela; Hwang, Wei-Ting; Levine, Bruce L.; June, Carl H.

    2014-01-01

    BACKGROUND CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe. METHODS We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (−1.81 cells per day) was significantly less than the decline in unmodified cells (−7.25 cells per day) (P = 0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National

  9. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.

    PubMed

    Tebas, Pablo; Stein, David; Tang, Winson W; Frank, Ian; Wang, Shelley Q; Lee, Gary; Spratt, S Kaye; Surosky, Richard T; Giedlin, Martin A; Nichol, Geoff; Holmes, Michael C; Gregory, Philip D; Ando, Dale G; Kalos, Michael; Collman, Ronald G; Binder-Scholl, Gwendolyn; Plesa, Gabriela; Hwang, Wei-Ting; Levine, Bruce L; June, Carl H

    2014-03-06

    CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others

  10. Blood doping: potential of blood and urine sampling to detect autologous transfusion.

    PubMed

    Segura, J; Lundby, C

    2014-05-01

    The collection of blood, its storage as red blood cell (RBC) concentrates and its reinjection is prohibited; until now, the practice cannot be reliably detected. A recent innovation-the haematological module of the athlete's biological passport-can provide authorities with indications towards autologous blood transfusion. In situations where a given athlete has been exposed to altitude, heat stress, sickness, etc, additional evidence may be needed to establish beyond any reasonable doubt that a blood transfusion may actually have occurred. Additional evidence may be obtained from at least three different approaches using parameters related to blood and urine matrices.Genomics applied to mRNA or miRNA is one of the most promising analytical tools. Proteomics of changes associated with RBC membranes may reveal the presence of cells stored for some time, as can an abnormal pattern of size distribution of aged cells. In urine, high concentrations of metabolites of plasticisers originating from the blood storing bags strongly suggest a recent blood transfusion. We emphasise the usefulness of simultaneously obtaining and then analysing blood and urine for complementary evidence of autologous blood transfusion ('blood doping').

  11. Photoporation and cell transfection using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  12. Nanothermite-Based Microsystem for Drug Delivery and Cell Transfection

    DTIC Science & Technology

    2008-12-01

    presented. 2. EXPERIMENTAL 2.1 Nanothermite Preparation Nanothermite mixtures consisting of Bi2O3 nanoparticles and Al nanoparticles were used...for the transfection experiments reported herein. The Bi2O3 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the...particles were physically mixed in isopropanol (IPA) using ultrasonic agitation. Batches were mixed by dispersing 200mg of Bi2O3 in 1.5 mL of IPA by

  13. Uptake of DNA by cancer cells without a transfection reagent.

    PubMed

    Kong, Yanping; Zhang, Xianbo; Zhao, Yongliang; Xue, Yanfang; Zhang, Ye

    2017-01-21

    Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting

  14. Enhanced transfection of brain tumor suppressor genes by photochemical internalization

    NASA Astrophysics Data System (ADS)

    Chou, Chih H.; Sun, Chung-Ho; Zhou, Yi-Hong; Madsen, Steen J.; Hirschberg, Henry

    2011-03-01

    One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

  15. [Transfected miR-1908 inhibits renal fibrosis via targeting transforming growth factor beta 1].

    PubMed

    Xie, Fei; Li, Xiaoshun; Wei, Chan; Gou, Liang; Dang, Yanlong; Shan, Zelei

    2015-12-01

    To explore the regulatory role of miR-1908 in renal fibrosis. The level of miR-1908 and transforming growth factor beta 1 (TGF-β1) mRNA during renal fibrosis were detected with real-time quantitative PCR. Bioinformatics and luciferase reporter gene analyses were applied to determine the targeting relationship between miR-1908 and TGF-β1 mRNA. After primary human renal interstitial fibroblasts were transfected with miR-1908 adenoviral expression vector in vitro, Western blotting was used to detect the protein levels of TGF-β1, smad2/3 and matrix metalloproteinase 2 (MMP-2) in the cells. Six weeks after intraperitoneal injection of miR-1908 adenoviral vector, the renal tissue sections of the renal fibrosis mouse models were stained with Masson staining. Human miR-1908 showed a gradually decreasing expression during renal fibrosis process, which was completely contrary to the changes of TGF-β1 mRNA. Overexpression of miR-1908 suppressed the expressions of TGF-β1, smad2/3 and MMP-2 in human primary renal interstitial cells. The renal fibrosis was significantly relieved in the mice injected with miR-1908 adenovirus vector injection compared with the ones without injection. miR-1908 could inhibit renal fibrosis through targeting TGF-β1.

  16. Towards gene therapy based on femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Antkowiak, M.; Torres-Mapa, M. L.; McGinty, J.; Chahine, M.; Bugeon, L.; Rose, A.; Finn, A.; Moleirinho, S.; Okuse, K.; Dallman, M.; French, P.; Harding, S. E.; Reynolds, P.; Gunn-Moore, F.; Dholakia, K.

    2012-06-01

    Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

  17. Graphene and carbon nanotube nanocomposite for gene transfection.

    PubMed

    Hollanda, L M; Lobo, A O; Lancellotti, M; Berni, E; Corat, E J; Zanin, H

    2014-06-01

    Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

  18. Application of nanostructured biochips for efficient cell transfection microarrays

    NASA Astrophysics Data System (ADS)

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

    2007-01-01

    Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

  19. Suppression of RNA interference on expression of c-myc of SKOV3 ovarian carcinoma cell line.

    PubMed

    Ai, Z-H; Wang, J; Xu, Y-L; Zhu, X-L; Teng, Y-C

    2013-11-01

    To investigate suppression of RNA interference (RNAi) on expression of c-myc of SKOV3 ovarian carcinoma cell line. The c-myc -siRNA was designed and synthesized, then transfected to SKOV3 ovarian carcinoma cell lines. The cell lines were divided into four groups, including the blank control group, the siRNA transfection group, the mock transfection group and the negative control group. The expression level of c-myc mRNA and protein were detected by RT-PCR and Western blotting, respectively. The growth and proliferation of SKOV3 ovarian carcinoma cell lines were observed with CCK-8 assay. After transfected with c-myc -siRNA, the expression level of c-myc mRNA and protein were down-regulated, the growth and proliferation of SKOV3 ovarian carcinoma cell line were inhibited in the siRNA transfection group. There were significant differences between the siRNA transfection group and the blank control group (p < 0.05). The silencing efficiency was 77.78%, the protein suppression rate was 67.78%, and the inhibition ratio was 56.35% by CCK-8 assay in siRNA transfection group. The down-regulation of c-myc expression of SKOV3 ovarian carcinoma cell line by c-myc -siRNA can lead to the suppression of cancer cell proliferation. The small interfering RNAs technique can inhibit the proliferation of carcinoma cell by oncogene silencing.

  20. Osteoarthritic articular chondrocytes stimulate autologous T cell responses in vitro.

    PubMed

    Sakata, M; Masuko-Hongo, K; Nakamura, H; Onuma, H; Tsuruha, J I; Aoki, H; Nishioka, K; Kato, T

    2003-01-01

    To clarify the presence of specific T cell immune response to autologous chondrocytes in patients with osteoarthritis (OA). Peripheral blood mononuclear cells obtained from OA or post-traumatic patients were co-cultured with irradiated autologous chondrocytes, and their proliferative response was assessed using 3H-thymidine incorporation. Expression of HLA-class II molecules was also assessed on chondrocytes by immunohistochemistry or flow cytometry. T cell responses to autologous chondrocytes in OA yielded a significantly greater mean stimulation index (6.35 +/- 1.63) compared to controls (1.21 +/- 0.09, p < 0.01). This response was partially blocked by antibodies against HLA class I, class II, CD4 or CD8. Increased expression of HLA-DP, -DQ, and -DR was observed. This study showed the autologous T cell-stimulating property of OA chondrocytes in vitro. The elucidation of the autoimmune responses may contribute to the understanding of immune-mediated mechanisms in OA.

  1. Acute mental change and hemiplegia after autologous fat injection.

    PubMed

    Kang, Jun Ho; Park, Kyung Hye; Park, Jung Soo

    2016-11-01

    Autologous fat injection is a common procedure used for skin augmentation. It is known to be safe and simple, but severe complications have been reported at times. The authors observed a patient with acute large cerebral infarction including the territories of the anterior and middle cerebral arteries and optic nerve infarction developing after autologous fat transplantation. A 32-year-old woman was referred to the emergency room of our hospital due to sudden stupor. Thirty minutes earlier, she was undergoing cosmetic autologous fat injection into the glabella area by a plastic surgeon at a private clinic. The cause was confirmed to be anterior and middle cerebral arteries infarction on brain imaging studies. When a patient presents abrupt mental change, hemiplegia, ocular pain, or blindness after autologous fat particle injection, physicians must consider cerebral infarction and combined retinal artery occlusion.

  2. Autologous anti-metatype immune response in rabbits.

    PubMed

    Voss, E W; Moore, J K; Weidner-McGufficke, K M; Denzin, L K; Bedzyk, W D; Voss, V H

    1992-02-01

    Rabbits hyperimmunized with fluorescyl-conjugated KLH exhibited bound ligand associated with a high affinity circulating IgG anti-fluorescein population. After cessation of immunogen administration the liganded complexes were eventually spontaneously cleared from the circulation. Individual rabbits synthesized autologous anti-metatype antibodies specific for ligand-antibody complexes. Autologous anti-metatype antibodies reacted optimally with autologous liganded anti-fluorescein antibodies. However, cross reactivity was noted with allogenic rabbit liganded antibodies from three affinity-purified pools. An autologous anti-metatype response, reminiscent of autoanti-idiotype responses, has important implications concerning in vivo clearance of antigen-antibody complexes and may serve as a model to study immune complex diseases.

  3. Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy In Vivo.

    PubMed

    Do, Hai Van Thi; Loke, Wan Ting; Kee, Irene; Liang, Vivienne; David, Sebastian J; Gan, Shu Uin; Lee, Sze Sing; Ng, Wai Har; Koong, Heng Nung; Ong, Hock Soo; Lee, Kok Onn; Calne, Roy Y; Kon, Oi Lian

    2015-01-01

    Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire-Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >10(10) cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous β-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal

  4. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines.

    PubMed

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K

    2013-01-30

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [(3)H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [(3)H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis

    PubMed Central

    Behrens, Frank; Himsel, Andrea; Rehart, Stefan; Stanczyk, Joanna; Beutel, Björn; Zimmermann, Stefanie Y; Koehl, Ulrike; Möller, Burkhard; Gay, Steffen; Kaltwasser, Joachim P; Pfeilschifter, Josef M; Radeke, Heinfried H

    2007-01-01

    Objectives Regulatory T cells (Tregs) exert their anti‐inflammatory activity predominantly by cell contact‐dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA). Methods 44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ‐secreting cells was quantified in synovial tissue cell cultures, CD3‐depleted synovial tissue cell cultures, synovial tissue cultures co‐cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real‐time PCR for CD3ε, T‐bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein. Results RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co‐culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T‐bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity. Conclusion This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments. PMID:17392348

  6. Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation.

    PubMed

    Oishi, Isao

    2010-01-01

    Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.

  7. Collision of millimetre droplets induces DNA and protein transfection into cells

    PubMed Central

    Ikemoto, Kazuto; Sakata, Ichiro; Sakai, Takafumi

    2012-01-01

    Nonperturbing and simple transfection methods are important for modern techniques used in biotechnology. Recently, we reported that electrospraying can be applied to DNA transfection in cell lines, bacteria, and chicken embryos. However, the transfection efficiency was only about 2%. To improve the transfection rate, physical properties of the sprayed droplets were studied in different variations of the method. We describe a highly efficient technique (30–93%) for introduction of materials such as DNA and protein into living cells by electrospraying droplets of a high conductivity liquid onto cells incubated with the material for transfection. Electric conductivity has a sizable influence on the success of transfection. In contrast, molecular weight of the transfected material, types of ions in the electrospray solution, and the osmotic pressure do not influence transfection efficiency. The physical analysis revealed that collision of cells with millimetre-sized droplets activates intracellular uptake. PMID:22375250

  8. Generation of infectious RNA complexes in orbiviruses: RNA-RNA interactions of genomic segments

    PubMed Central

    Fajardo, Teodoro; AlShaikhahmed, Kinda; Roy, Polly

    2016-01-01

    Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of H Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules. PMID:27736800

  9. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    PubMed

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation.

  10. Gene transfection mediated by polyethyleneimine-polyethylene glycol nanocarrier prevents cisplatin-induced spiral ganglion cell damage

    PubMed Central

    Chen, Guan-gui; Mao, Min; Qiu, Li-zi; Liu, Qi-ming

    2015-01-01

    Polyethyleneimine-polyethylene glycol (PEI-PEG), a novel nanocarrier, has been used for transfection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein (XIAP) in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats, via scala tympani fenestration, before daily cisplatin injections. Auditory brainstem reflex tests demonstrated the protective effects of XIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels of XIAP mRNA expression in the cochlea. The present findings suggest that PEI-PEG nanocarrier-mediated XIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing. PMID:25878591

  11. Magnetic Tweezers-based 3D Microchannel Electroporation for High-Throughput Gene Transfection in Living Cells

    PubMed Central

    Liao, Wei-Ching; Chiang, Chi-Ling; Gallego-Perez, Daniel; Yang, Zhaogang; Lu, Wu; Byrd, John C.; Muthusamy, Natarajan; Lee, L. James.; Sooryakumar, Ratnasingham

    2015-01-01

    We report a novel, high throughput magnetic-tweezers based 3D microchannel electroporation system capable of transfecting 40,000 cells/cm2 on a single-chip for gene therapy, regenerative medicine and intracellular detection of target mRNA for screening cellular heterogeneity. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (> 90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum based approach, is significantly gentler on the cellular membrane yielding > 90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon was delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells. PMID:25469659

  12. Autologous fat grafting: use of closed syringe microcannula system for enhanced autologous structural grafting

    PubMed Central

    Alexander, Robert W; Harrell, David B

    2013-01-01

    Objectives Provide background for use of acquiring autologous adipose tissue as a tissue graft and source of adult progenitor cells for use in cosmetic plastic surgery. Discuss the background and mechanisms of action of closed syringe vacuum lipoaspiration, with emphasis on accessing adipose-derived mesenchymal/stromal cells and the stromal vascular fraction (SVF) for use in aesthetic, structural reconstruction and regenerative applications. Explain a proven protocol for acquiring high-quality autologous fat grafts (AFG) with use of disposable, microcannula systems. Design Explain the components and advantage of use of the patented super luer-lock and microcannulas system for use with the closed-syringe system. A sequential explanation of equipment selection for minimally traumatic lipoaspiration in small volumes is presented, including use of blunt injection cannulas to reduce risk of embolism. Results Thousands of AFG have proven safe and efficacious for lipoaspiration techniques for large and small structural fat grafting procedures. The importance and advantages of gentle harvesting of the adipose tissue complex has become very clear in the past 5 years. The closed-syringe system offers a minimally invasive, gentle system with which to mobilize subdermal fat tissues in a suspension form. Resulting total nuclear counting of undifferentiated cells of the adipose-derived -SVF suggests that the yield achieved is better than use of always-on, constant mechanical pump applied vacuum systems. Conclusion Use of a closed-syringe lipoaspiration system featuring disposable microcannulas offers a safe and effective means of harvesting small volumes of nonmanipulated adipose tissues and its accompanying progenitor cells within the SVF. Closed syringes and microcannulas are available as safe, sterile, disposable, compact systems for acquiring high-quality AFG. Presented is a detailed, step-by-step, proven protocol for performing quality autologous structural adipose

  13. Evaluation of autologous bone marrow in wound healing in animal model: a possible application of autologous stem cells.

    PubMed

    Akela, Ashok; Nandi, Samit Kumar; Banerjee, Dibyajyoti; Das, Partha; Roy, Subhasis; Joardar, Siddhartha Narayan; Mandal, Mohan; Das, Pradip Kumar; Pradhan, Nisith Ranjan

    2012-10-01

    A study was conducted to evaluate the potential of autologous bone marrow-derived cells in comparison with buffy coat of autologous blood for rapid cutaneous wound healing in rabbit model. Three square full-thickness skin excisional wounds were created in 15 selected experimental animals (rabbit) divided randomly into three groups. The wound was treated with autologous bone marrow cells in plasma (group 1), buffy coat of blood in plasma (group 2) and autologous plasma as control (group 3). Wounds were observed for 30 days for granulation tissue formation, biochemical, histomorphological and histochemical evaluation. In this study, granulation tissue appeared significantly lesser in wounds of group 3 animals followed by group 2 and 1 animals. Neovascularisation, granulation tissue formation, denser, thicker and better arranged collagen fibres, reticulin fibres and elastin fibres formation was more in group 1 as compared with other groups. It was concluded that the application of bone marrow-derived nucleated cells into the wound margins resulted in early and significantly faster rate of complete healing as compared with buffy coat of autologous blood and autologous plasma (control). This approach may be beneficial in various surface wounds that heal at a slower rate and recommended for healing of various complicated wound in future.

  14. Hormonal induction of transfected genes depends on DNA topology.

    PubMed Central

    Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

    1990-01-01

    Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors. Images PMID:2153920

  15. Tissue Engineering Using Transfected Growth-Factor Genes

    NASA Technical Reports Server (NTRS)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  16. Antitumor effects obtained by autologous Lewis lung cancer cell vaccine engineered to secrete mouse interleukin 27 by means of cationic liposome.

    PubMed

    Zhang, Junfeng; Tian, Hongwei; Li, Can; Cheng, Lin; Zhang, Shuang; Zhang, Xiaomei; Wang, Ruibo; Xu, Fen; Dai, Lei; Shi, Gang; Chen, Xiaolei; Li, Yiming; Du, Tao; Deng, Jie; Liu, Yu; Yang, Yang; Wei, Yuquan; Deng, Hongxin

    2013-10-01

    Interleukin-27 (IL-27), a novel IL-6/IL-12 family cytokine, plays an important role in the early regulation of Th1 responses. The cytokine IL-27 can exert a variety of immune-regulatory functions including cytotoxic T lymphocyte (CTL), CD4+, CD8+ T lymphocytes activation and interferon-γ (IFN-γ) production. In this study, we developed an effective and gene modified tumor cell vaccine. Lewis lung cancer cell LL/2 transfected with the DOTAP:cholesterol cationic liposome could express the mouse IL-27 (mIL-27) gene at a relative high level. The resultant transfectants were then irradiated with X-ray and used as a tumor cell vaccine. This tumor cell vaccine not only contained tumor associated antigen (TAA) of LL/2 cells but also secreted mIL-27 which could induce immune response in mice. The mice vaccinated with LL/2-mIL-27 performed strong tumor inhibiting effect accompanied with a high IFN-γ production. Both CD4+ and CD8+ T lymphocytes were significantly elevated in these mice vaccinated with LL/2-mIL-27 cell vaccine. Moreover, after depletion of CD4+, CD8+ T lymphocytes by injection of antibodies against CD4 and CD8, the vaccinated mice inoculated with autologous LL/2 cells were not protected from tumor challenge. In contrast, vaccinated mice inoculated with autologous LL/2 cells were treated with antibody against natural killer (NK)cells or normal rat IgG still possessed strong antitumor activity. Our data suggested that DOTAP:cholesterol cationic liposome was quite useful in generating an autologous tumor cell vaccine and mIL-27 could be therapeutically used to potentiate the host antitumor immunity.

  17. Transient and stable transfection in the protozoan parasite Entamoeba invadens

    PubMed Central

    Ehrenkaufer, Gretchen M.; Singh, Upinder

    2012-01-01

    Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development. PMID:22561071

  18. Graphene for improved femtosecond laser based pluripotent stem cell transfection.

    PubMed

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Khanyile, Thulile; Warner, Jamie H

    2014-05-01

    Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples

  19. [Preserving autologous heart operation for dilated cardiomyopathy].

    PubMed

    Hoshino, Joji; Fukada, Yasuhisa; Hirota, Masanori; Kondo, Taichi; Isomura, Tadashi

    2013-01-01

    We report non transplant surgical procedure (preserving autologous heart operation) for the patients with dilated cardiomyopathy( DCM), clinical outcomes, and the factor of predict prognosis. Since May 2000, 258 patients received surgical procedure for 11 years. We performed mitral surgery (plasty or replacement) for the patients with more than mild mitral regurgitation (MR). We performed papirally muscule plication since 2005, and we performed 2nd chordal cutting since 2008, for the patients with MR due to mitral tethering. The surgical left ventricular reconstruction( SVR) was performed for the patients with dilated left ventricular. We use spackle tracking echocardiography to decide the type of SVR since 2008. Hospital death was 18.2%, and late cardiac death was 27.5%.Almost the cause of death was congestive heart failure and ventricular arrhythmia. Five years survival was 58%, 10 years survival was 39%. Preoperative condition, emergent operation, inotropic support, intra aortic balloon pumping(IABP),affect the prognosis. But left ventricular size did not affect it. Surgical treatment for the patient with DCM should be performed with stable preoperative condition.

  20. Autologous hematopoietic cell transplantation in multiple sclerosis.

    PubMed

    Bell, Simon M; Sharrack, Basil; Snowden, John A

    2017-01-01

    Autologous haematopoietic cell transplantation (AHCT) is an evolving treatment avenue in multiple sclerosis (MS), which may be highly effective in controlling disease activity and improving disability. However, AHCT is associated with intrinsic toxicities and risks compared with conventional therapies. With growing experience in patient selection and treatment delivery, AHCT is increasingly considered an option in patients with aggressive disease that's responding poorly to disease modifying therapy. Areas covered: This article provides an introduction to AHCT and looks at its development as a treatment for MS over the last 20 years. It also highlights potential mechanisms of action, patient selection, and future trends for this treatment approach. Expert opinion: Currently published data suggest that AHCT's use is associated with significant reduction in MS disease activity and marked improvement in disability when used in patients with highly active relapsing remitting disease. Its long term safety and efficacy have not been fully evaluated but as increasing clinical trial data are published, its use is likely to grow. Further randomised controlled studies are needed to compare AHCT with standard disease modifying therapies and to optimise transplant regimens. Mechanistic studies may provide potential markers for response and a better understanding of disease pathogenesis.

  1. Adhesive strength of autologous fibrin glue.

    PubMed

    Yoshida, H; Hirozane, K; Kamiya, A

    2000-03-01

    To establish an easy and rapid method for measuring the adhesive strength of fibrin glue and to clarify the factor(s) most affecting the strength, a study was made on the effect of the concentration of plasma components on the strength of cryoprecipitate (Cryo) prepared from a subject's own autologous plasma to be used as fibrin glue. The adhesive strength of the Cryo was measured with various supporting materials instead of animal skin using a tester of tension and compression. The results were as follows: (1) the strength of Cryo applied to ground flat glass (4 cm2) was significantly greater than that applied to clear glass, clear plastic, or smooth and flat wood chips; (2) the adhesive strength of Cryo depended on the concentration of thrombin with the optimal concentration being 50 units/ml; (3) the concentration of CaCl2 did not affect the adhesive strength of Cryo; (4) the adhesive reaction was dependent on the temperature and the adhesive strength more quickly reached a steady state at 37 degrees C than at lower temperature; (5) the adhesive strength was correlated well with the total concentration of fibrinogen and fibronectin. These results indicate that the adhesive strength of Cryo can be easily and quickly evaluated using a tester and ground glass with thrombin at 50 units/ml, and that the adhesive strength of Cryo can be predicted from the total concentration of fibrinogen and fibronectin.

  2. Autologous cord blood transplantation for metastatic neuroblastoma.

    PubMed

    Ning, Botao; Cheuk, Daniel Ka-Leung; Chiang, Alan Kwok-Shing; Lee, Pamela Pui-Wah; Ha, Shau-Yin; Chan, Godfrey Chi-Fung

    2016-03-01

    Auto-SCT is a common approach for metastatic neuroblastoma with the intention to rescue hematopoiesis after megadose chemotherapy. PBSC or BM is the usual stem cell source for auto-SCT. Auto-CBT for neuroblastoma has very rarely been performed. Currently, case reports are available for two patients only. We performed 13 auto-SCTs for high-risk neuroblastoma from 2007 to 2013, including four cases of metastatic neuroblastoma aged 11-64 months treated with auto-CBT. All four patients had partial or CR to upfront treatments before auto-CBT. Nucleated cell dose and CD34+ cell dose infused were 2.8-8.7 × 10(7) /kg and 0.36-3.9 × 10(5) /kg, respectively. Post-thawed viability was 57-76%. Neutrophil engraftment (>0.5 × 10(9) /L) occurred at 15-33 days, while platelet engraftment occurred at 31-43 days (>20 × 10(9) /L) and 33-65 days (>50 × 10(9) /L) post-transplant, respectively. There was no severe acute or chronic complication. Three patients survived for 1.9-7.7 yr without evidence of recurrence. One patient relapsed at 16 months post-transplant and died of progressive disease. Cord blood may be a feasible alternative stem cell source for auto-SCT in patients with stage 4 neuroblastoma, and outcomes may be improved compared to autologous PBSC or BM transplants.

  3. Clinical outcomes following osteochondral autologous transplantation (OATS).

    PubMed

    Lahav, Amit; Burks, Robert T; Greis, Patrick E; Chapman, Andrew W; Ford, Gregory M; Fink, Barbara P

    2006-07-01

    This study evaluated the clinical outcome in 21 patients (22 knees) undergoing osteochondral autologous transplantation (OATS) in the knee over a 5-year period. Sixteen knees in 15 patients were available for follow-up at an average of 40 months after the procedure. The clinical outcome was analyzed using the IKDC and Knee and Osteoarthritis Outcome Score (KOOS) evaluation forms, a subjective questionnaire, and a clinical examination. At final follow-up, the average KOOS result for pain was 80.6 (range: 56-94), symptoms 53.6 (range: 25-71), function of activities of daily living 93.4 (range: 79-100), function of sports and recreational activities 65.3 (range: 20-100), and quality of life 51.0 (range: 6-88). The average IKDC score was 68.2. On our subjective questionnaire, the average preoperative grade given was 3.1 (range: 1-7) with an improvement at the most recent follow-up to a grade of 8.0 (range: 5-10) (P < .00001). Thirteen (86%) patients reported that they would have the surgery again if they had to make the decision a second time. Age did not correlate with subjective results on the IKDC evaluation (P = .7048) or score difference on our questionnaire (P = .9175). This procedure provides an option for articular resurfacing of the femoral condyles for focal areas of chondral defects with promising results regarding subjective improvement.

  4. Transfection of chondromodulin I into human breast cancer cells and its effect on the inhibition of cancer cell growth.

    PubMed

    Shao, Jie; Gan, Linghong; Wang, Jie

    2016-05-01

    Breast cancer affects one in every eight women, and has been associated with higher rates of female mortality than any other cancer type, with the exception of lung cancer. It has been reported that chondromodulin I (ChM-I) was able to suppress tumor angiogenesis and growth in vivo. In order to investigate the antitumor action of ChM‑I on human breast cancer cells, a plasmid expressing ChM‑I was constructed and transfected into human breast cancer cells using an adenoviral vector. Reverse transcription‑polymerase chain reaction detected ChM‑I expression in human breast cancer cell lines, whereas no expression was detected in the control groups. In order to assess the effect of ChM‑I on human breast cancer cells, cell counting kit‑8 (CCK‑8) and colony formation analyses were used to detect tumor cell proliferation, and the proliferation of ChM‑I‑transfected cells was significantly reduced, as compared with the control. In addition, the mRNA expression levels of cell cycle‑associated genes in ChM‑I‑transfected cells were significantly decreased, as compared with the control, which suggested that ChM‑I transfection was able to inhibit the expression of genes associated with the cell cycle. The results of the present study indicated that ChM‑I was able to inhibit the growth of breast cancer cells; thus suggesting that ChM-I may have potential clinical applications in the treatment of breast cancer.

  5. 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

  6. 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.

    PubMed

    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

    2016-04-28

    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

  7. Allogeneic versus autologous blood transfusion and survival after radical prostatectomy

    PubMed Central

    Chalfin, Heather J.; Frank, Steven M.; Feng, Zhaoyong; Trock, Bruce J.; Drake, Charles G.; Partin, Alan W.; Humphreys, Elizabeth; Ness, Paul M.; Jeong, Byong C.; Lee, Seung B.; Han, Misop

    2016-01-01

    BACKGROUND Potential adverse effects of blood transfusion (BT) remain controversial, especially for clinical outcomes after curative cancer surgery. Some postulate that immune modulation after allogeneic BT predisposes to recurrence and death, but autologous superiority is not established. This study assessed whether BT is associated with long-term prostate cancer recurrence and survival a large single-institutional radical prostatectomy (RP) database. STUDY DESIGN AND METHODS Between 1994 and 2012, a total of 11,680 patients had RP with available outcome and transfusion data. A total of 7443 (64%) had complete covariate data. Clinical variables associated with biochemical recurrence-free survival (BRFS), cancer-specific survival (CSS), and overall survival (OS) were identified with Cox proportional hazards models for three groups: no BT (reference, 27.7%, n = 2061), autologous BT only (68.8%, n = 5124), and any allogeneic BT (with or without autologous, 3.5%, n = 258). RESULTS Median (range) follow-up was 6 (1–18) years. Kaplan-Meier analysis showed significantly decreased OS (but not BRFS or PCSS) in the allogeneic group versus autologous and no BT groups (p = 0.006). With univariate analysis, any allogeneic BT had a hazard ratio (HR) of 2.29 (range, 1.52–3.46; p < 0.0001) for OS, whereas autologous BT was not significant (HR, 1.04 [range, 0.82–1.32], p = 0.752). In multivariable models, neither autologous nor allogeneic BT was independently associated with BRFS, CSS, or OS, and a dose response was not observed for allogeneic units and BRFS. CONCLUSION Although allogeneic but not autologous BT was associated with decreased long-term OS, after adjustment for confounding clinical variables, BT was not independently associated with OS, BRFS, or CSS regardless of transfusion type. Notably, no association was observed between allogeneic BT and cancer recurrence. Observed differences in OS may reflect confounding. PMID:24601996

  8. Intraoperative hemodilution and autologous platelet rich plasma collection: two techniques for collecting fresh autologous blood.

    PubMed

    Triulzi, D J; Ness, P M

    1995-03-01

    Intraoperative hemodilution (IH) and autologous platelet rich plasma (APRP) collection are two techniques used to obtain autologous blood in the operating room. They have been used to reduce allogeneic blood exposure in patients undergoing both cardiac and non-cardiac surgery. Both components have the advantage of providing fresh blood not subject to the storage lesion. Whole blood (IH) or platelet rich plasma is removed from the patient as anesthesia is induced and replaced with acellular fluid. The blood is transfused back after bypass or major bleeding has ceased. Although used commonly, the data supporting the use of either technique are controversial. Methodologic problems which have confounded studies evaluating their utility include: poorly defined transfusion criteria, concommitant use of other blood conservation techniques (i.e. cell salvage, pharmacologic agents, hypothermia, controlled hypotension) and changing transfusion practices with greater tolerance of normovolemic anemia. Randomized controlled studies with well defined up to date transfusion criteria are needed to identify patients likely to benefit from these techniques.

  9. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    PubMed

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  10. Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

    PubMed

    Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

    2006-11-01

    Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

  11. DNA photo-oxidative damage hazard in transfection complexes.

    PubMed

    Rudiuk, Sergii; Franceschi-Messant, Sophie; Chouini-Lalanne, Nadia; Perez, Emile; Rico-Lattes, Isabelle

    2011-01-01

    Complexes of DNA with various cationic vectors have been largely used for nonviral transfection, and yet the photochemical stability of DNA in such complexes has never been considered. We studied, for the first time, the influence of DNA complexation by a cationic lipid and polymers on the amount of damage induced by benzophenone photosensitization. The localization of benzophenone inside the hydrophobic domains formed by a cationic lipid, DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and close to DNA, locally increases the photoinduced cleavage by the reactive oxygen species generated. The same effect was found in the case of DNA complexation with an amphiphilic polymer (polynorbornenemethyleneammonium chloride). However, a decrease in DNA damage was observed in the case of complexation with a hydrophilic polymer (polyethylenimine). The DNA protection in this case was because of the absence of benzophenone hydrophobic incorporation into the complex, and to DNA compaction which decreased the probability of radical attack. These results underline the importance of the chemical structure of the nonviral transfection vector in limiting the risks of photo-oxidative damage of the complexed DNA. © 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.

  12. Flow-type electroporation chips for gene transfection

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Jen, Chung-Min; Huang, Ming-Yuan; Lin, Xi-Zhang

    2000-08-01

    Electroporation is a technique with which DNA molecules can be delivered into cells in a chamber using high electric field pulses. The limited amount of target cells and the potential risk from the high voltage are the two drawbacks in this technique. This study aimed to fabricate an electroporation chip to manage large amount of cells continuously with a lower applied voltage. The electroporation chip, consisting of a micro-channel with thin film electrodes made of gold or platinum wire electrodes on both sides, was fabricated on PMMA material using evaporation, photolithography, wet- etching, lift-off, and fusion-bonding methods. The suspension fluid of Huh-7 cell lines (1 x 106 cells/ml) mixed with 10 micrometers plasmids equipped with lacZ genes in a volume of 500 (mu) l flowed through the channel with a variety of flow rates under a series of square pulses. The transfection rate was evaluated with blue-staining cells under X-Gal stain 24 hours later. The dimensions of the channel were 5 mm wide, 0.2 mm high, and 25 mm long. Two types of electrodes, parallel-plate type and parallel-line type electrodes, were fabricated and tested in these experiments. The fabricated microchip can deliver genes into the flowing of cells. The electric pulse frequency that determines the shock number for each cell for a fixed flow rate can be optimized for better transfection and survival rates.

  13. Towards optical cell transfection inside a micro flow cell

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2014-03-01

    For optical transfection, cells are shortly subjected to intense, focused laser radiation which leads to a temporary opening in the cell membrane. Although the method is very efficient and ensures high cell viability, the targeting of single cells with laser pulses is a tedious and slow approach. We present first measurements aiming at an experimental setup which is suitable for high throughput and automated optical cell transfection. In our setup, cells flow through a micro flow cell where they are spatially confined. The laser radiation is focused into the cell in a way that an elongated focal region is realized. This makes the time consuming aiming of the laser beam at individual cells unnecessary and opens the possibility to develop a completely automated system. The elongated laser focal region is realized by a quasi-Bessel beam which is generated by an axicon lens setup and continuously scanned from side to side of the cell. We present test measurements of the newly employed setup and discuss its suitability to be fully integrated into a flow cell sequencing system.

  14. Multiple C4/Slp genes distinguished by expression after transfection.

    PubMed Central

    Robins, D M; Malissen, M; Hood, L; Ferreira, A; Walthall, D; Mitchell, M

    1986-01-01

    The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes. Images PMID:3023818

  15. [Enhanced liposomal transfection through the application of sex steroids in gynecological cancer cells].

    PubMed

    Köster, F; Finas, D; Saupp, A; Schulz, C; Diedrich, K; Hauser, C; Felberbaum, R

    2003-01-01

    Liposomal transfection in gene therapeutic application against gynecological malignoma does not reach satisfying efficacy. A desirable goal would be the specific intensification of transfection in these kind of cells. Steroids have successfully been used in other systems to increase liposomal transfection and hopefully there might be a specific impact of sexual steroids in cells from high sex steroid receptor expressing malignoma, like some mamma- and endometrium cancer. The mamma carcinoma cell line T-47D was transfected with the transfection agent DOTAP and cyclodextrin solubilized steroids and cholesterol were co-applied. The efficiency of transfection was followed by luciferase activity resulting from the transfected reporter gene. Like cholesterol, which is already established as transfection co-agent, also the steroids progesterone, estrogen, testosterone and hydrocortisone provoked a clear increase in transfection efficiency shown in a dose dependent manner. These results indicate the usefulness of steroids as additives for liposomal transfection procedures in gene therapeutic application. As sexual steroid receptors migrate into the nucleus of a cell after binding its specific ligand a targeted enhancement of transfection is supposable in malignoma overexpressing steroid receptors. There is evidence that plasmid DNA can be co-transported with nuclear proteins into the nucleus.

  16. Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080).

    PubMed Central

    Fertala, A; Sieron, A L; Ganguly, A; Li, S W; Ala-Kokko, L; Anumula, K R; Prockop, D J

    1994-01-01

    Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy

  17. Genetically modified mesenchymal stem/stromal cells transfected with adiponectin gene can stably secrete adiponectin.

    PubMed

    Hossain, Md Murad; Murali, Malliga Raman; Kamarul, Tunku

    2017-08-01

    Mesenchymal stem/stromal cells (MSCs) hold promises for the treatment of diverse diseases and regeneration of injured tissues. Genetic modification of MSCs through gene delivery might enhance their therapeutic potential. Adiponectin has been appeared as a potential biomarker for predicting various diseases. Plasma adiponectin levels are negatively correlated with various metabolic and vascular diseases and supplementation of exogenous adiponectin ameliorates the diseases. This study aims to develop adiponectin secreting genetically modified MSCs (GM-MSCs) as a potent strategic tool to complement endogenous adiponectin for the treatment of adiponectin deficiency diseases. Human bone marrow derived MSCs were isolated, expanded in vitro and transfected with adiponectin gene containing plasmid vector. Total RNA was extracted and cDNA was prepared by reverse transcription polymerase chain reaction (RT-PCR). The expression of adiponectin gene and protein in GM-MSCs was analyzed by PCR and Western blotting respectively. The secretion of adiponectin protein from GM-MSCs was analyzed by enzyme-linked immunosorbent assay. The expression of adiponectin gene and plasmid DNA was detected in GM-MSCs but not in control group of MSCs. Adiponectin gene expression was detected in GM-MSCs at 2, 7, 14, 21 and 28days after transfection. Western blotting analysis revealed the expression of adiponectin protein only in GM-MSCs. The GM-MSCs stably secreted adiponectin protein into culture media at least for 4weeks. GM-MSCs express and secret adiponectin protein. Therefore, these adiponectin secreting GM-MSCs could be instrumental for the supplementation of adiponectin in the treatment of adiponectin deficiency related diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration.

    PubMed

    Chung, Ji Hyung; Im, Eun Kyoung; Jin, Tae Won; Lee, Seung-Min; Kim, Soo Hyuk; Choi, Eun Young; Shin, Min-Jeong; Lee, Kyung Hye; Jang, Yangsoo

    2011-04-30

    Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

  19. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

    PubMed

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-24

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  20. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-01

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  1. Whole blood cells loaded with messenger RNA as an anti-tumor vaccine.

    PubMed

    Phua, Kyle K L; Boczkowski, David; Dannull, Jens; Pruitt, Scott; Leong, Kam W; Nair, Smita K

    2014-06-01

    The use of a cell-based vaccine composed of autologous whole blood cells loaded with mRNA is described. Mice immunized with whole blood cells loaded with mRNA encoding antigen develop anti-tumor immunity comparable to DC-RNA immunization. This approach offers a simple and affordable alternative to RNA-based cellular therapy by circumventing complex, laborious and expensive ex vivo manipulations required for DC-based immunizations.

  2. Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

    PubMed Central

    Tcherepanova, Irina; Harris, Jason; Starr, Aijing; Cleveland, Jaclyn; Ketteringham, Helen; Calderhead, David; Horvatinovich, Joe; Healey, Don; Nicolette, Charles A.

    2008-01-01

    Background Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. Methodology/Principal Findings The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. Conclusion/Significance This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral

  3. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    PubMed Central

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  4. Transfection with liver-type glutaminase cDNA alters gene expression and reduces survival, migration and proliferation of T98G glioma cells.

    PubMed

    Szeliga, Monika; Obara-Michlewska, Marta; Matyja, Ewa; Łazarczyk, Marzena; Lobo, Carolina; Hilgier, Wojciech; Alonso, Francisco J; Márquez, Javier; Albrecht, Jan

    2009-07-01

    Liver-type glutaminase (LGA) is a glutaminase isoform that has been implicated in transcription modulation. LGA mRNA is absent from postoperative samples of primary gliomas and is low in cultured astrocytes. In this study, stable transfection of T98G cells with a vector carrying human LGA sequence increased the expression of LGA mRNA and protein, and the ability of the cells to degrade glutamine (Gln), as manifested by a three-fold reduction of their steady-state Gln content and a 2.5-fold increase of their glutamate (Glu) content. The transfected cells (TLGA cells) showed a 40% decrease of cell survival as assessed by colony formation, well correlated with significant reduction of mitochondrial activity as demonstrated with MTT test. Also, a 45% reduction of cell migration and a 47% decrease of proliferation index (Ki67 immunostaining) were found as compared with sham-transfected cells. Microarray analysis, which included over 47,000 transcripts, revealed a significantly altered expression of 85 genes in TLGA, but not in sham-transfected or control cells (P < 0.005). Microarray data were confirmed with real-time PCR analysis for eight genes potentially relevant to malignancy: S100A16, CAPN2, FNDC3B, DYNC1LI1, TIMP4, MGMT, ADM, and TIMP1. Of these changes, decreased expression of S100A16 and MGMT can be best reconciled with the current views on the role of their protein products in glioma malignancy. Malignancy-reducing effect of newly inserted LGA mRNA in glioblastoma cells can be reconciled with a hypothesis that absence of such a modulatory mechanism in glia-derived tumors deprived of LGA mRNA may facilitate some aspects of their progression.

  5. Cryopreservation of Autologous Blood (Red Blood Cells, Platelets and Plasma)

    NASA Astrophysics Data System (ADS)

    Ebine, Kunio

    Prevention of post-transfusion hepatitis is still a problem in cardiovascular surgery. We initiated the cryopreservation of autologous blood for the transfusion in elective cardiovascular surgery since 1981. This study includes 152 surgical cases in which autologous frozen, allogeneic frozen, and/or allogeneic non-frozen blood were used. In the 152 surgical cases, there were 69 cases in which autologous blood only (Group I) was used; 12 cases with autologous and allogeneic frozen blood (Group II); 46 cases with autologous and allgeneic frozen plus allogeneic non-frozen blood (Group III); and 25 cases with allogeneic frozen plus allogeneic non-frozen blood (Group IV). No hepatitis developed in Groups I (0%) and II (0%), but there was positive hepatitis in Groups III (4.3%) and IV (8.0%) . In 357 cases of those who underwent surgery with allogeneic non-frozen whole blood during the same period, the incidence rate of hepatitis was 13.7% (49/357). Patients awaiting elective surgery can store their own blood in the frozen state. Patients who undergo surgery with the cryoautotransfusion will not produce any infections or immunologic reactions as opposed to those who undergo surgery with the allogeneic non-frozen blood.

  6. Orthopaedic-induced anemia: the fallacy of autologous donation programs.

    PubMed

    Cushner, Fred D; Hawes, Thomas; Kessler, Debra; Hill, Kala; Scuderi, Giles R

    2005-02-01

    Total knee arthroplasty is associated with significant blood loss. Despite the initiation of various blood conservation modalities, allogeneic transfusion has yet to be eliminated. One hundred forty-eight patients who had unilateral primary total knee arthroplasties during a 3-year period were evaluated retrospectively for blood loss and transfusion rates. The patients were prescribed one unit of preoperative autologous donation that was to be transfused automatically on postoperative Day 1. Allogeneic transfusion was based on symptoms, and no numerical transfusion triggers were used. The preoperative autologous donation program resulted in increased preoperative anemia. Whereas only 26.2% of patients were in the high transfusion-risk group (hemoglobin >10 g/dL and < or = 13 g/dL) before surgery, 55.7% of patients were in this high-risk category after preoperative autologous donation. The patients did not recover from the autologous donations that occurred 4 weeks before surgery. A mean hemoglobin level of 14.0 g/dL was seen before donation, whereas the mean preoperative hemoglobin level decreased to 12.6 g/dL. We think that a preoperative autologous donation program leads to an increased risk of anemia before surgery.

  7. Autologous chondrocyte implantation: superior biologic properties of hyaline cartilage repairs.

    PubMed

    Henderson, Ian; Lavigne, Patrick; Valenzuela, Herminio; Oakes, Barry

    2007-02-01

    Information regarding the quality of autologous chondrocyte implantation repair is needed to determine whether the current autologous chondrocyte implantation surgical technology and the subsequent biologic repair processes are capable of reliably forming durable hyaline or hyaline-like cartilage in vivo. We report and analyze the properties and qualities of autologous chondrocyte implantation repairs. We evaluated 66 autologous chondrocyte implantation repairs in 57 patients, 55 of whom had histology, indentometry, and International Cartilage Repair Society repair scoring at reoperation for mechanical symptoms or pain. International Knee Documentation Committee scores were used to address clinical outcome. Maximum stiffness, normalized stiffness, and International Cartilage Repair Society repair scoring were higher for hyaline articular cartilage repairs compared with fibrocartilage, with no difference in clinical outcome. Reoperations revealed 32 macroscopically abnormal repairs (Group B) and 23 knees with normal-looking repairs in which symptoms leading to arthroscopy were accounted for by other joint disorders (Group A). In Group A, 65% of repairs were either hyaline or hyaline-like cartilage compared with 28% in Group B. Autologous chondrocyte repairs composed of fibrocartilage showed more morphologic abnormalities and became symptomatic earlier than hyaline or hyaline-like cartilage repairs. The hyaline articular cartilage repairs had biomechanical properties comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs.

  8. Autologous hematopoietic stem cell transplantation for systemic sclerosis.

    PubMed

    Milanetti, Francesca; Bucha, Jurate; Testori, Alessandro; Burt, Richard K

    2011-03-01

    Systemic sclerosis is a rare disorder manifesting as skin and internal organ fibrosis, a diffuse vasculopathy, inflammation, and features of autoimmunity. Patients with diffuse cutaneous disease or internal organ involvement have a poor prognosis with high mortality. To date no therapy has been shown to reverse the natural course of the disease. Immune suppressive drugs are commonly utilized to treat patients, but randomized trials have generally failed to demonstrate any long-term benefit. In phase I/II trials, autologous hematopoietic stem cell transplantation (HSCT) has demonstrated impressive reversal of skin fibrosis, improved functionality and quality of life, and stabilization of internal organ function, but initial studies were complicated by significant treatment-related mortality. Treatment-related mortality was reduced by better pre-transplant evaluation to exclude patients with compromised cardiac function and by treating patients earlier in disease, allowing selected patients the option of autologous HSCT treatment. There are currently three ongoing randomized trials of autologous HSCT for systemic sclerosis: ASSIST (American Systemic Sclerosis Immune Suppression versus Transplant), SCOT (scleroderma cyclophosphamide versus Transplant), and ASTIS (Autologous Stem cell Transplantation International Scleroderma). The results from these trials should clarify the role of autologous HSCT in the currently limited therapeutic arsenal of severe systemic sclerosis.

  9. Calf Augmentation and Reshaping with Autologous Fat Grafting.

    PubMed

    Mundinger, Gerhard S; Vogel, James E

    2016-02-01

    Despite multiple advantages of fat grafting for calf augmentation and re-shaping over traditional silicone calf implants, few reports have been published. To report our technique and results with autologous fat grafting for calf augmentation and reshaping. A retrospective review of the senior author's (JEV) experience with autologous fat grafting for calf augmentation was performed. Medial and lateral calf augmentation was accomplished with injection of prepared autologous lipoaspirate intramuscularly and subcutaneously. Over a 5-year period, 13 patients underwent calf augmentation and reshaping with the described technique. Ten cases were bilateral (77%), and 3 cases (23%) were performed for congenital leg discrepancies. Mean 157 cc of prepared lipoaspirate was transferred per leg, with roughly 60% and 40% transferred into the medial and lateral calf, respectively. Four patients (31%) underwent a second round of autologous fat injection for further calf augmentation because they desired more volume. At mean 19.6 month follow-up, durable augmentation and improvement in calf contour was documented by comparison of standardized preoperative and postoperative photographs. Autologous calf fat grafting is a viable alternative to traditional implant-based calf augmentation for congenital calf discrepancies and the aesthetic pseudo-varus deformity. This technique provides results comparable to those obtainable with traditional methods. LEVEL OF EVIDENCE 4: Therapeutic. © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

  10. Autologous blood use in percutaneous nephrolithotomy.

    PubMed

    Stoller, M L; Lee, K L; Schwartz, B F; Viele, M K

    1999-09-01

    Preoperative autologous blood (AUB) donation has decreased patient exposure to allogenic blood (ALB) products and associated infectious risk. The risk of contracting hepatitis C and human immunodeficiency virus is 1 in 103,000 and 1 in 678,000, respectively, after receiving 1 U ALB. Elective surgical procedures require surgeons to offer preoperative AUB donation in California. Unused AUB is discarded. We report our use of AUB obtained for percutaneous nephrolithotomy. A retrospective study of 144 consecutive patients who underwent 193 percutaneous nephrolithotomies between January 1994 and April 1998 at one of four teaching hospitals at the University of California, San Francisco was performed. Preoperative AUB donation, transfusion rates, hemoglobin levels, blood use, and costs were analyzed. Ninety-six units of blood were collected from 63 patients (44%) and were available for 70 procedures (36%). The overall transfusion rate per procedure was 7%, with 13 patients receiving a total of 24 U, 7 AUB and 17 ALB. Eighty-nine units (92.7%) of AUB were discarded, and the transfusion rate in donors and nondonors was similar. There was no significant difference in preoperative hemoglobin or operative blood loss between donors and nondonors. The 13 transfused patients had a lower preoperative hemoglobin ( 11.5 versus 12.8 g/dL; P = 0.029) and higher operative blood loss as measured by hemoglobin level (3.2 versus 1.6 g/dL; P <0.001). Blood bank charges for ALB and AUB were $ 119/U and $244 to $498/U, respectively, depending on transportation and thawing charges. Routine preoperative blood donation adds substantial cost for minimal benefit, given the low infectious risk of ALB and the two- to fourfold higher cost of AUB. In our series, women had an increased incidence of blood transfusion compared with men. AUB donation may provide peace of mind but is rarely used and is discarded 93% of the time.

  11. Adult Human Olfactory Epithelial-Derived Progenitors: A Potential Autologous Source for Cell-Based Treatment for Parkinson's Disease

    PubMed Central

    Wang, Meng; Lu, Chengliang

    2012-01-01

    Human adult olfactory epithelial-derived neural progenitors (hONPs) can differentiate along several neural lineages in response to morphogenic signals in vitro. A previous study optimized the transfection paradigm for the differentiation of hONPs to dopaminergic neurons. This study engrafted cells modified by the most efficient transfection paradigm for dopaminergic neural restriction and pretransfected controls into a unilateral neurotoxin, 6-hydroxydopamine-induced parkinsonian rat model. Approximately 35% of the animals engrafted with hONPs had improved behavioral recovery as demonstrated by the amphetamine-induced rotation test, as well as a corner preference and cylinder paw preference, over a period of 24 weeks. The pre- and post-transfected groups produced equivalent responses, indicating that the toxic host environment supported hONP dopaminergic differentiation in situ. Human fibroblasts used as a cellular control did not diminish the parkinsonian rotational deficits at any point during the study. Increased numbers of tyrosine hydroxylase (TH)-positive cells were detected in the engrafted brains compared with the fibroblast-implanted and medium-only controls. Engrafted TH-positive hONPs were detected for a minimum of 6 months in vivo; they were multipolar, had long processes, and migrated beyond their initial injection sites. Higher dopamine levels were detected in the striatum of behaviorally improved animals than in equivalent regions of their nonrecovered counterparts. Throughout these experiments, no evidence of tumorigenicity was observed. These results support our hypothesis that human adult olfactory epithelial-derived progenitors represent a unique autologous cell type with promising potential for future use in a cell-based therapy for patients with Parkinson's disease. PMID:23197853

  12. Protease-sensitive transfection of Bacillus subtilis with bacteriophage GA-1 DNA: a probable case of heterologous transfection.

    PubMed

    Arwert, F; Venema, G

    1974-03-01

    The host bacterium of bacteriophage GA-1, Bacillus sp. G1R, was compared with respect to its taxonomic relationship to Bacillus subtilis, B. licheniformis, and B. pumilis. The physiological-biochemical properties of Bacillus sp. G1R are equal to those of B. licheniformis, but the thermal denaturation midpoint of G1R DNA differs by 3 C and the buoyant density by 0.005 g/cm(3) from that of B. licheniformis. Transformation with G1R donor DNA was neither observed in B. licheniformis nor in B. subtilis-competent recipients. Bacteriophage GA-1 shows neither infectivity on B. licheniformis nor on B. subtilis. However, infection of competent B. subtilis cultures with phenol-extracted GA-1 DNA results in the production of infective GA-1 particles. The transfecting activity of GA-1 DNA is destroyed by treatment with proteolytic enzymes. Resistance of transfecting DNA to inactivation by trypsin develops earlier than that to inactivation by DNase. Protease-treated GA-1 DNA competes with transforming DNA to approximately the same extent as does untreated GA-1 DNA, suggesting that uptake of GA-1 DNA is not affected by protease treatment. CsCl density gradient centrifugation reveals that the density of trypsinized GA-1 DNA is 0.004 g/cm(3) greater than that of untreated DNA.

  13. Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA Targeting Col1a1 for Hydrogel-Based Tissue-Engineered Cartilage

    PubMed Central

    Perrier-Groult, Emeline; Pasdeloup, Marielle; Malbouyres, Marilyne; Galéra, Philippe

    2013-01-01

    Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-β1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-β1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-β1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte–agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 siRNA

  14. Mouse in utero electroporation: controlled spatiotemporal gene transfection.

    PubMed

    Matsui, Asuka; Yoshida, Aya C; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi

    2011-08-15

    In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early

  15. Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

    PubMed Central

    Matsui, Asuka; Yoshida, Aya C.; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi

    2011-01-01

    In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in

  16. A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo

    NASA Astrophysics Data System (ADS)

    Zhao, Deyao; Huang, Dong; Li, Yang; Wu, Mengxi; Zhong, Wenfeng; Cheng, Qiang; Wang, Xiaoxia; Wu, Yidi; Zhou, Xiao; Wei, Zewen; Li, Zhihong; Liang, Zicai

    2016-01-01

    Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.

  17. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    PubMed

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization.

  18. Improving diagnosis of appendicitis. Early autologous leukocyte scanning.

    PubMed

    DeLaney, A R; Raviola, C A; Weber, P N; McDonald, P T; Navarro, D A; Jasko, I

    1989-10-01

    A prospective nonrandomized study investigating the accuracy and utility of autologous leukocyte scanning in the diagnosis of apendicitis was performed. One hundred patients in whom the clinical diagnosis of appendicitis was uncertain underwent indium 111 oxyquinoline labelling of autologous leukocytes and underwent scanning 2 hours following reinjection. Of 32 patients with proved appendicitis, three scans revealed normal results (false-negative rate, 0.09). Of 68 patients without appendicitis, three scans had positive results (false-positive rate, 0.03; sensitivity, 0.91; specificity, 0.97; predictive value of positive scan, 0.94; predictive value of negative scan, 0.96; and overall accuracy, 0.95). Scan results altered clinical decisions in 19 patients. In 13 cases, the scan produced images consistent with diagnoses other than appendicitis, expediting appropriate management. Early-imaging111 In oxyquinoline autologous leukocyte scanning is a practical and highly accurate adjunct for diagnosing appendicitis.

  19. Autologous hematopoietic stem cell transplantation in progressive severe multiple sclerosis.

    PubMed

    Pandit, Awadh Kishor; Prasad, Kameshwar; Seth, Tulika

    2015-01-01

    Multiple sclerosis (MS) is a chronic inflammatory disease of central nervous system (CNS), which is disabling and majorly involves younger population. Various available treatments in forms of immunomodulation are not very effective; however, stem cell transplantation seems to be promising in recent literature. The current case report is a novel evidence for autologous hematopoietic stem cell transplantation (HSCT) in progressive MS. A 33 year old male with secondary progressive MS (SPMS), after being failed and/or intolerance to standard approved interferon (IFN) and mitoxantrone therapy, autologous HSCT was administered. At 2years of post-stem cell transplantation follow-up, he has remained stable with some improvement in functional status (Expanded Disability Status Scale (EDSS) reduced by 1.5), with no relapse, no treatment related complications, and no fresh magnetic resonance imaging (MRI) lesions. Autologous stem cell transplantation may be beneficial in progressive forms of MS, but needs to be tested in well-designed randomized trial.

  20. Therapeutic potential of autologous stem cell transplantation for cerebral palsy.

    PubMed

    Purandare, Chaitanya; Shitole, D G; Belle, Vaijayantee; Kedari, Aarti; Bora, Neeta; Joshi, Meghnad

    2012-01-01

    Background. Cerebral palsy (CP) is a severe disabling disease with worldwide incidence being 2 to 3 per 1000 live births. CP was considered as a noncurable, nonreparative disorder, but stem cell therapy offers a potential treatment for CP. Objective. The present study evaluates the safety and efficacy of autologous bone-marrow-derived mononuclear cell (BMMNCs) transplantation in CP patient. Material and Methods. In the present study, five infusions of autologous stem cells were injected intrathecally. Changes in neurological deficits and improvements in function were assessed using Gross Motor Function Classification System (GMFCS-E&R) scale. Results. Significant motor, sensory, cognitive, and speech improvements were observed. Bowel and bladder control has been achieved. On the GMFCS-E&R level, the patient was promoted from grade III to I. Conclusion. In this study, we report that intrathecal infusion of autologous BMMNCs seems to be feasible, effective, and safe with encouraging functional outcome improvements in CP patient.

  1. [Treatment of relapsed Hodgkin lymphoma after autologous stem cell transplantation].

    PubMed

    Illés, Árpád; Simon, Zsófia; Udvardy, Miklós; Magyari, Ferenc; Jóna, Ádám; Miltényi, Zsófia

    2017-08-01

    Approximately 10-30% of Hodgkin lymphoma patients relapses or experience refractory disease after first line treatment. Nowadays, autologous stem cell transplantation can successfully salvage half of these patients, median overall survival is only 2-2.5 years. Several prognostic factors determine success of autologous stem cell transplantation. Result of transplantation can be improved considering these factors and using consolidation treatment, if necessary. Patients who relapse after autologous transplantation had worse prognosis, treatment of this patient population is unmet clinical need. Several new treatment options became available in the recent years (brentuximab vedotin and immuncheckpoint inhibitors). These new treatment options offer more chance for cure in relapsed/refractory Hodgkin patients. Outcome of allogenic stem cell transplantation can be improved by using haploidentical donors. New therapeutic options will be discussed in this review. Orv Hetil. 2017; 158(34): 1338-1345.

  2. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  3. Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

    NASA Astrophysics Data System (ADS)

    McBain, S. C.; Griesenbach, U.; Xenariou, S.; Keramane, A.; Batich, C. D.; Alton, E. W. F. W.; Dobson, J.

    2008-10-01

    Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection—rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.

  4. 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.

  5. Clickable Poly(ionic liquids): A Materials Platform for Transfection.

    PubMed

    Freyer, Jessica L; Brucks, Spencer D; Gobieski, Graham S; Russell, Sebastian T; Yozwiak, Carrie E; Sun, Mengzhen; Chen, Zhixing; Jiang, Yivan; Bandar, Jeffrey S; Stockwell, Brent R; Lambert, Tristan H; Campos, Luis M

    2016-09-26

    The potential applications of cationic poly(ionic liquids) range from medicine to energy storage, and the development of efficient synthetic strategies to target innovative cationic building blocks is an important goal. A post-polymerization click reaction is reported that provides facile access to trisaminocyclopropenium (TAC) ion-functionalized macromolecules of various architectures, which are the first class of polyelectrolytes that bear a formal charge on carbon. Quantitative conversions of polymers comprising pendant or main-chain secondary amines were observed for an array of TAC derivatives in three hours using near equimolar quantities of cyclopropenium chlorides. The resulting TAC polymers are biocompatible and efficient transfection agents. This robust, efficient, and orthogonal click reaction of an ionic liquid, which we term ClickabIL, allows straightforward screening of polymeric TAC derivatives. This platform provides a modular route to synthesize and study various properties of novel TAC-based polymers.

  6. Transient and stable transfection in the protozoan parasite Entamoeba invadens.

    PubMed

    Ehrenkaufer, Gretchen M; Singh, Upinder

    2012-07-01

    Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Integrated optical transfection system using a microlens fiber combined with microfluidic gene delivery

    PubMed Central

    Ma, N.; Ashok, P. C.; Stevenson, D. J.; Gunn-Moore, F. J.; Dholakia, K.

    2010-01-01

    Optical transfection is a promising technique for the delivery of foreign genetic material into cells by transiently changing the permeability of the cell membrane. Of the different optical light sources that have been used, femtosecond laser based transfection has been one of the most effective methods for optical transfection which is generally implemented using a free space bulk optical setup. In conventional optical transfection methods the foreign genetic material to be transfected is homogenously mixed in the medium. Here we report the first realization of an integrated optical transfection system which can achieve transfection along with localized drug delivery by combining a microlens fiber based optical transfection system with a micro-capillary based microfluidic system. A fiber based illumination system is also incorporated in the system in order to achieve visual identification of the cell boundaries during transfection. A novel fabrication method is devised to obtain easy and inexpensive fabrication of microlensed fibers, which can be used for femtosecond optical transfection. This fabrication method offers the flexibility to fabricate a microlens which can focus ultra-short laser pulses at a near infrared wavelength to a small focal spot (~3 µm) whilst keeping a relatively large working distance (~20 µm). The transfection efficiency of the integrated system with localized plasmid DNA delivery, is approximately 50%, and is therefore comparable to that of a standard free space transfection system. Also the use of integrated system for localized gene delivery resulted in a reduction of the required amount of DNA for transfection. The miniaturized, integrated design opens a range of exciting experimental possibilities, including the dosing of tissue slices, targeted drug delivery, and targeted gene therapy in vivo. PMID:21258501

  8. DNA-poly(diallyldimethylammonium chloride) complexation and transfection efficiency.

    PubMed

    Alatorre-Meda, Manuel; Taboada, Pablo; Krajewska, Barbara; Willemeit, Markus; Deml, Alexander; Klösel, Roland; Rodríguez, Julio R

    2010-07-29

    The present work assesses the influence of the cationic charge density (CD) and the cationic valence of poly(diallyldimethylammonium chloride) (pDADMAC) on the DNA compaction and subsequent transfection. Four homopolymers (CD = 1, with different valences) and one copolymer, poly(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (CD < 1, equivalent in valence to one of the homopolymers), were studied. The characterization of the DNA-pDADMAC complexes (polyplexes) as a function of the polycation nitrogen to DNA phosphate molar ratios, N/P, was done by means of conductometry, electrophoretic mobility (zeta-potential), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), atomic force microscopy (AFM), and beta-galactosidase (ONPG) and luciferase expression assays at 25 degrees C and physiological pH. In general, all polyplexes rendered compact and stable structures (R(H) approximately 100 nm) with positive surface charges ( approximately 11 mV) but low transfection efficiencies. As revealed by ITC, the DNA-pDADMAC complexation was characterized by a high binding affinity, the process being entropically driven. In particular, two characteristic ratios ((N/P)c and (N/P)*) were detected. Conductometry and ITC data demonstrated that the DNA compaction ratio, (N/P)c, was mainly governed by CD. Meanwhile the ratio from which the polyplex size remained constant, (N/P)*, was found to be valence-dependent as revealed by DLS. On the other hand, the low transfer rate of the polyplexes appeared to be correlated with the high binding affinity observed throughout the complexation process and with a core-shell structure the complexes presumably adopt.

  9. Monetite granules versus particulate autologous bone in bone regeneration.

    PubMed

    Torres, Jesús; Tamimi, Iskandar; Cabrejos-Azama, Jatsue; Tresguerres, Isabel; Alkhraisat, Mohammad; López-Cabarcos, Enrique; Hernández, Gonzalo; Tamimi, Faleh

    2015-07-01

    The aim of this study was to test bone tissue response to monetite granules in comparison with intramembranous autologous bone graft in a rabbit calvaria critical size defect model. Novel monetite granules were synthesized by thermal conversion of set brushite cement. Eight female New Zealand rabbits were used for this study. Two identical 10mm diameter bicortical cranial defects were created in each animal. One of the defects was grafted with monetite granules while the contralateral was grafted with granules of intramembranous autologous bone as control. Animals were sacrificed 8 weeks after surgery and biopsies were taken for histological and histomorphometrical evaluation under light microscopy. Wilcoxon test was used for statistical analysis. The bone defects treated with either autologous bone or monetite granules were able to heal within the study period. Upon histological observation the defects treated with autologous bone granules resembled the adjacent intact calvaria, whereas the defects treated with monetite showed a high infiltration of new bone and only 13.4±8.4% of remaining granules. The percentage of bone volume in the defects of the control group (71±9%) was 16% higher than in the study group (55±10%) (p<0.05). The percentage of augmented mineralized tissue volume in the study group (68±18%) was not significantly different from the control group (p>0.05). The amount of augmented mineralized tissue in the bone defects obtained with monetite granules was not significantly different from that obtained with autologous bone. This study confirms the potential of monetite based biomaterials as an alternative to autologous bone graft. Copyright © 2015 Elsevier GmbH. All rights reserved.

  10. [Cell-based therapy options for osteochondral defects. Autologous mesenchymal stem cells compared to autologous chondrocytes].

    PubMed

    Grässel, S; Anders, S

    2012-05-01

    Cartilage defects are multifactorial and site-specific and therefore need a clear analysis of the underlying pathology as well as an individualized therapy so that cartilage repair lacks a one-for-all therapy. The results of comparative clinical studies using cultured chondrocytes in autologous chondrocyte implantation (ACI) have shown some superiority over conventional microfracturing under defined conditions, especially for medium or large defects and in long-term durability. Adult mesenchymal stem cells can be isolated from bone marrow, have the potency to proliferate in culture and are capable of differentiating into the chondrogenic pathway. They represent a promising versatile cell source for cartilage repair but the ideal conditions for cultivation and application in cartilage repair are not yet known or have not yet been characterized. Adding a scaffold offers mechanical stability and advances chondrogenic differentiation for both possible cell sources.

  11. Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

    PubMed

    Kachalaki, Saeed; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi; Shanehbandi, Dariush; Mohammadinejad, Sina; Mansoori, Behzad

    2015-10-01

    Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells. Copyright © 2015. Published by Elsevier Masson SAS.

  12. Preoperative autologous plateletpheresis in patients undergoing open heart surgery.

    PubMed

    Tomar, Akhlesh S; Tempe, Deepak K; Banerjee, Amit; Hegde, Radhesh; Cooper, Andrea; Khanna, S K

    2003-07-01

    Blood conservation is an important aspect of care provided to the patients undergoing cardiac operations with cardiopulmonary bypass (CPB). It is even more important in patients with anticipated prolonged CPB, redo cardiac surgery, patients having negative blood group and in patients undergoing emergency cardiac surgery. In prolonged CPB the blood is subjected to more destruction of important coagulation factors, in redo surgery the separation of adhesions leads to increased bleeding and difficulty in achieving the haemostasis and in patients with negative blood group and emergency operations, the availability of sufficient blood can be a problem. Harvesting the autologous platelet rich plasma (PRP) can be a useful method of blood conservation in these patients. The above four categories of patients were prospectively studied, using either autologous whole blood donation or autologous platelet rich plasma (PRP) harvest in the immediate pre-bypass period. Forty two patients were included in the study and randomly divided into two equal groups of 21 each, control group (Group I) in which one unit of whole blood was withdrawn, and PRP group (Group II) where autologous plateletpheresis was utilised. After reversal of heparin, autologous whole blood was transfused in the control group and autologous PRP was transfused in the PRP group. The chest tube drainage and the requirement of homologous blood and blood products were recorded. Average PRP harvest was 643.33 +/- 133.51 mL in PRP group and the mean whole blood donation was 333.75 +/- 79.58 mL in the control group. Demographic, preoperative and intra operative data showed no statistically significant differences between the two groups. The PRP group patients drained 26.44% less (p<0.001) and required 38.5% less homologous blood and blood products (p<0.05), in the postoperative period. Haemoglobin levels on day zero (day of operation) and day three were statistically not different between the two groups. We conclude

  13. Necrobiotic xanthogranuloma successfully treated with autologous stem cell transplantation.

    PubMed

    Goede, Jeroen S; Misselwitz, Benjamin; Taverna, Christian; Schanz, Urs; Dispenzieri, Angela; Hummel, Yvonne; Trüeb, Ralph M; Fehr, Jörg

    2007-04-01

    Paraproteinemia can be complicated by necrobiotic xanthogranuloma. Therapeutic options for this progressive disease are limited, and there is no agreement on a single best strategy. We report the case of a patient with a massive periorbital infiltration narrowing the palpebral fissure and blinding the patient. Conventional myeloma therapy had only limited benefit in our patient. However, he was successfully treated with high-dose chemotherapy followed by autologous stem cell transplantation, rendering the patient free of symptoms. This is the first report of autologous stem cell transplantation in a patient with necrobiotic xanthogranuloma.

  14. An assessment of the overexpression of BMP-2 in transfected human osteoblast cells stimulated by mineral trioxide aggregate and Biodentine.

    PubMed

    Rodrigues, E M; Gomes-Cornélio, A L; Soares-Costa, A; Salles, L P; Velayutham, M; Rossa-Junior, C; Guerreiro-Tanomaru, J M; Tanomaru-Filho, M

    2017-01-21

    To evaluate the effect of MTA and Biodentine on viability, osteogenic differentiation and BMP-2 expression in osteogenic cells. Saos-2 cells were used as a model of osteoblastic cells. Overexpression of BMP-2 was induced by transfection of a CMV-driven plasmid construct including the human BMP-2 coding sequence, and stably transfected cells were selected. Cell viability was assessed by the mitochondrial dehydrogenase enzymatic (MTT) assay. The bioactivity of the materials was evaluated by the alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2 and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). Viability tests revealed that MTA and Biodentine were not cytotoxic at the higher dilution (1 : 8) to BMP-2-transfected cells. MTA and Biodentine exhibited the highest ALP activity when compared to the Saos-BMP-2-unexposed control group (P < 0.05). Cell exposure to Biodentine and MTA had a significant stimulatory effect on the formation of mineralized nodules (P < 0.05). The highest increase in BMP-2 gene expression was observed after 3 days of BMP-2-transfected cells exposure to MTA and Biodentine in non-osteogenic medium in relation to Saos-BMP-2-unexposed control cells (P < 0.05). Exposure of cells to MTA in osteogenic medium for 1 day increased ALP gene expression by approximately 1.3-fold in relation to Saos-BMP-2-unexposed control cells (P < 0.05). Both MTA and Biodentine showed biocompatibility and bioactivity in Saos-BMP-2 overexpressing cells. Biodentine had a significantly greater effect on mineralization than MTA. Both MTA and Biodentine enhanced BMP-2 mRNA expression in the transfected system. Both MTA and Biodentine are suitable materials to improve osteoblastic cell mineralization. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  15. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

    PubMed

    Chettab, Kamel; Roux, Stéphanie; Mathé, Doriane; Cros-Perrial, Emeline; Lafond, Maxime; Lafon, Cyril; Dumontet, Charles; Mestas, Jean-Louis

    2015-01-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

  16. DNA repair investigations using siRNA.

    PubMed

    Miller, Holly; Grollman, Arthur P

    2003-06-11

    Small interfering RNA (siRNA) is a revolutionary tool for the experimental modulation of gene expression, in many cases making redundant the need for specific gene mutations and allowing examination of the effect of modulating essential genes. It has now been shown that siRNA phenotypes resulting from stable transfection with short hairpin RNA (shRNA) can be transmitted through the mouse germ line and Rosenquist and his colleagues have used shRNA, which is processed in vivo to siRNA, to create germline transgenic mice in which a target DNA repair gene has been silenced. Here, Holly Miller and Arthur P. Grollman give the background of these discoveries, provide an overview of current uses, and look at future applications of this research.

  17. Application of fluorescence spectroscopy and multispectral imaging for non-invasive estimation of GFP transfection efficiency

    NASA Astrophysics Data System (ADS)

    Tamošiūnas, M.; Jakovels, D.; Lihačovs, A.; Kilikevičius, A.; Baltušnikas, J.; Kadikis, R.; Šatkauskas, S.

    2014-10-01

    Electroporation and ultrasound induced sonoporation has been showed to induce plasmid DNA transfection to the mice tibialis cranialis muscle. It offers new prospects for gene therapy and cancer treatment. However, numerous experimental data are still needed to deliver the plausible explanation of the mechanisms governing DNA electro- or sono-transfection, as well as to provide the updates on transfection protocols for transfection efficiency increase. In this study we aimed to apply non-invasive optical diagnostic methods for the real time evaluation of GFP transfection levels at the reduced costs for experimental apparatus and animal consumption. Our experimental set-up allowed monitoring of GFP levels in live mice tibialis cranialis muscle and provided the parameters for DNA transfection efficiency determination.

  18. Oscillating Magnet Array−Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies

    PubMed Central

    Subramanian, Mahendran; Tyler, Aimee-Jayne; Luther, Eva Maria; Daniel, Elena Di; Lim, Jenson; Dobson, Jon

    2017-01-01

    To develop treatments for neurodegenerative disorders, it is critical to understand the biology and function of neurons in both normal and diseased states. Molecular studies of neurons involve the delivery of small biomolecules into cultured neurons via transfection to study genetic variants. However, as cultured primary neurons are sensitive to temperature change, stress, and shifts in pH, these factors make biomolecule delivery difficult, particularly non-viral delivery. Herein we used oscillating nanomagnetic gene transfection to successfully transfect SH-SY5Y cells as well as primary hippocampal and cortical neurons on different days in vitro. This novel technique has been used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability. From these observations and other related studies, we suggest that oscillating nanomagnetic gene transfection is an effective method for gene delivery into hard-to-transfect neuronal cell types. PMID:28336862

  19. Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro

    PubMed Central

    MA, XUEXIAO; LIN, YAZHOU; YANG, KUN; YUE, BIN; XIANG, HONGFEI; CHEN, BOHUA

    2015-01-01

    Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)-mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV-mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT-qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase-3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV-mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase-3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV-mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive

  20. Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells

    PubMed Central

    FENG, YUANYUAN; LUO, SHOUMING; YANG, PAN; SONG, ZHIYUAN

    2016-01-01

    The ‘funny’ current, also known as the If current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The If current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional If channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the If current reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the If current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was −101.2±4.6 mV and the time constant of activation was 324±41 msec under −160 mV. In the group B cells the If current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to −92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of HCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed If channels

  1. Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells.

    PubMed

    Feng, Yuanyuan; Luo, Shouming; Yang, Pan; Song, Zhiyuan

    2016-04-01

    The 'funny' current, also known as the If current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The If current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional If channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the If current reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the If current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was -101.2±4.6 mV and the time constant of activation was 324±41 msec under -160 mV. In the group B cells the If current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to -92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of HCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed If channels may have a

  2. Autologous serum eye drops for dry eye

    PubMed Central

    Pan, Qing; Angelina, Adla; Zambrano, Andrea; Marrone, Michael; Stark, Walter J; Heflin, Thomas; Tang, Li; Akpek, Esen K

    2014-01-01

    Background Theoretically, autologous serum eye drops (AS) have a potential advantage over traditional therapies based on the assumption that AS serve not only as a lacrimal substitute to provide lubrication, but also contain other biochemical components mimicking natural tears more closely. The application of AS in dry eye treatment has gained popularity as a second-line therapy in the treatment of dry eye. Published studies on the subject indicate that autologous serum could be an effective treatment for dry eye. Objectives To evaluate the efficacy and safety of AS compared to artificial tears for treating dry eye. Search methods We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2013, Issue 3), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLD MEDLINE, (January 1950 to April 2013), EMBASE (January 1980 to April 2013), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to April 2013), the meta Register of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We also searched the Science Citation Index Expanded database (September 2013) and reference lists of included studies. We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 15 April 2013. Selection criteria We included randomized controlled trials (RCTs) in which AS was compared to artificial tears in the treatment of dry eye in adults. Data collection and analysis Two review authors independently screened all titles and abstracts and assessed full-text articles of potentially eligible trials. Two review authors extracted data and assessed the methodological quality and characteristics of the included trials.We contacted investigators for missing data

  3. Autologous serum eye drops for dry eye.

    PubMed

    Pan, Qing; Angelina, Adla; Marrone, Michael; Stark, Walter J; Akpek, Esen K

    2017-02-28

    Theoretically, autologous serum eye drops (AS) offer a potential advantage over traditional therapies on the assumption that AS not only serve as a lacrimal substitute to provide lubrication but contain other biochemical components that allow them to mimic natural tears more closely. Application of AS has gained popularity as second-line therapy for patients with dry eye. Published studies on this subject indicate that autologous serum could be an effective treatment for dry eye. We conducted this review to evaluate the efficacy and safety of AS given alone or in combination with artificial tears as compared with artificial tears alone, saline, placebo, or no treatment for adults with dry eye. We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (2016, Issue 5), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2016), Embase (January 1980 to July 2016), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to July 2016), the ISRCTN registry (www.isrctn.com/editAdvancedSearch), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We also searched the Science Citation Index Expanded database (December 2016) and reference lists of included studies. We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 5 July 2016. We included randomized controlled trials (RCTs) that compared AS versus artificial tears for treatment of adults with dry eye. Two review authors independently screened all titles and abstracts and assessed full-text reports of potentially eligible trials. Two review authors extracted data and assessed risk of bias and characteristics of included trials. We contacted investigators to ask for missing data. For both primary and

  4. Autologous serum eye drops for dry eye.

    PubMed

    Pan, Qing; Angelina, Adla; Zambrano, Andrea; Marrone, Michael; Stark, Walter J; Heflin, Thomas; Tang, Li; Akpek, Esen K

    2013-08-27

    =Theoretically, autologous serum eye drops (AS) have a potential advantage over traditional therapies based on the assumption that ASserve not only as a lacrimal substitute to provide lubrication, but also contain other biochemical components mimicking natural tears more closely. The application of AS in dry eye treatment has gained popularity as a second-line therapy in the treatment of dry eye.Published studies on the subject indicate that autologous serum could be an effective treatment for dry eye. To evaluate the efficacy and safety of AS compared to artificial tears for treating dry eye. We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2013, Issue 3),Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE,(January 1950 to April 2013), EMBASE (January 1980 to April 2013), Latin American and Caribbean Literature on Health Sciences(LILACS) (January 1982 to April 2013), themetaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov(www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We also searched the Science Citation Index Expanded database (September 2013) and reference lists of included studies. We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 15 April 2013. We included randomized controlled trials (RCTs) in which AS was compared to artificial tears in the treatment of dry eye in adults. Two review authors independently screened all titles and abstracts and assessed full-text articles of potentially eligible trials. Two review authors extracted data and assessed the methodological quality and characteristics of the included trials.We contacted investigators for missing data. For both primary and secondary outcomes, we reported mean differences with corresponding 95

  5. Enhancement of gene silencing potency and nuclease stability by chemically modified duplex RNA.

    PubMed

    Kubo, Takanori; Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki

    2007-01-01

    In this study, we describe a development of chemically modified dsRNAs with improved biological properties. These dsRNAs possess an enhanced RNAi activity and a dramatically increased stability in cell cultured medium (containing 10% serum) in comparison with widely used 21nt siRNA. The chemically modified dsRNAs manifested a high longterm gene suppression after one week post-transfection, whereas 21nt siRNA showed a high RNAi activity only after 48 h cell transfection.

  6. 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.

  7. Effect of phosphatase and tensin homology deleted on chromosome 10 (PTEN) gene transfection on reversal of multidrug resistance in K562/ADM cells.

    PubMed

    Cheng, Zhiyong; Yang, Ning; Liang, Wentong; Yan, Xiaoyan; Li, Lin; Pan, Ling

    2012-07-01

    The phosphatase and tensin homology deleted on chromosome 10 (PTEN) gene is a novel tumor suppressor gene of the phosphatase family. Studies have shown that the PTEN gene is probably involved in human malignant disease pathogenesis, multidrug resistance, angiogenesis and extramedullary infiltration. This study was designed to investigate the effect of wild-type PTEN gene transfection on drug resistance reversal in K562/ADM leukemia cells in vitro and the possible mechanism. A recombinant adenovirus containing green fluorescent protein gene and wild-type PTEN gene (Ad-PTEN-GFP) or a recombined adenovirus containing green fluorescent protein gene only (Ad-GFP) was transfected into K562/ADM cells. These cells were then treated with different concentrations of adriamycin, cytarabine or arsenic trioxide, respectively. The half-maximal inhibitory concentration (IC(50)) of each drug was detected by MTT assay and the drug resistance reversal factor (RF) was calculated. The proliferation inhibition rate of these K562/ADM cells treated with or without the above-mentioned drugs was determined by MTT assay and the apoptosis rate was evaluated by flow cytometry. PTEN, nuclear factor-κB (NF-κB), I-κB, p53, multidrug resistance genes MDR1 and MRP, and apoptosis related genes Bcl-2, Bcl-xL and Bax mRNA levels were detected by real-time fluorescence relative-quantification reverse transcription polymerase chain reaction (FQ-PCR). PTEN, Akt, p-Akt and NF-κB (p65) protein levels were detected by Western blot. Results showed that PTEN gene transfection could increase the sensitivity of K562/ADM cells to chemotherapeutic drugs. The drug resistance reversal index of adriamycin, cytarabine and arsenic trioxide was 3.8-fold, 2.65-fold and 2.64-fold, respectively, after PTEN gene transfection. NF-κB, MDR1, Bcl-2 and Bcl-xL mRNA levels as well as p-Akt and NF-κB (p65) protein levels were down-regulated, while p53 and Bax mRNA levels were up-regulated in K562/ADM cells after

  8. Autologous buccal mucosa graft augmentation for foreshortened vagina.

    PubMed

    Grimsby, Gwen M; Bradshaw, Karen; Baker, Linda A

    2014-05-01

    Vaginal foreshortening after pelvic surgery or radiotherapy may lead to dyspareunia and decreased quality of life. Unfortunately, little literature exists regarding treatment options for this debilitating problem. Autologous buccal mucosal grafting has been previously reported for creation of a total neovagina and the repair of postvaginoplasty vaginal stenosis. Autologous buccal mucosa offers several advantages as a replacement material for vaginal reconstruction. Vaginal and oral buccal mucosa are both hairless, moist, nonkeratinized stratified squamous epithelia. Buccal mucosa has a dense layer of elastic fibers, imparting both elasticity and strength, and acquires a robust neovascularity with excellent graft take. The graft material is readily available and donor site scars are hidden in the mouth. A 60-year-old woman had vaginal foreshortening to 4.5 cm 15 years after radical hysterectomy and brachytherapy for endometrial cancer. She was unable to have intercourse despite attempted vaginal dilation. Her foreshortened vagina was successfully augmented with autologous buccal mucosa grafting at the apex, increasing her vaginal length to 8 cm and permitting pain-free intercourse. Even in the face of an altered surgical field after radical hysterectomy and radiation, autologous buccal mucosa is an option for vaginal reconstruction for vaginal foreshortening.

  9. Autologous epidermal cell suspension: A promising treatment for chronic wounds.

    PubMed

    Zhao, Hongliang; Chen, Yan; Zhang, Cuiping; Fu, Xiaobing

    2016-02-01

    Chronic wounds have become an increasing medical and economic problem of aging societies because they are difficult to manage. Skin grafting is an important treatment method for chronic wounds, which are refractory to conservative therapy. The technique involving epidermal cell suspensions was invented to enable the possibility of treating larger wounds with only a small piece of donor skin. Both uncultured and cultured autologous epidermal cell suspensions can be prepared and survive permanently on the wound bed. A systematic search was conducted of EMBASE, Cochrane Library, PubMed and web of science by using Boolean search terms, from the establishment of the database until May 31, 2014. The bibliographies of all retrieved articles in English were searched. The search terms were: (epithelial cell suspension OR keratinocyte suspension) and chronic and wound. From the included, 6 studies are descriptive interventions and discussed the use of autologous keratinocyte suspension to treat 61 patients' chronic wound. The various methods of preparation of epidermal cell suspension are described. The advantages and shortcomings of different carriers for epidermal cell suspensions are also summarised. Both uncultured and cultured autologous epidermal cell suspensions have been used to treat chronic wounds. Although the limitations of these studies include the small number of patient populations with chronic wounds and many important problems that remain to be solved, autologous epidermal cell suspension is a promising treatment for chronic wounds. Copyright © 2015 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

  10. A Role For Photodynamic Therapy In Autologous Bone Marrow Transplantation

    NASA Astrophysics Data System (ADS)

    Sieber, Fritz

    1988-02-01

    Simultaneous exposure to the amphipathic fluorescent dye merocyanine 540 (MC 540) and light of a suitable wavelength rapidly kills leukemia, lymphoma, and neuroblastoma cells but spares normal pluripotent hematopoietic stem cells. Tests in several preclinical models and early results of a phase I clinical trial suggest that MC 540-mediated photosensitization may be useful for the extracorporeal purging of autologous remission bone marrow grafts.

  11. Autologous Bone Marrow Transplantation for Poor-Prognosis Neuroblastoma

    DTIC Science & Technology

    1987-01-01

    Recent pilot studies of intensive chemotherapy and total body irradiation (TBI) followed by allogeneic bone marrow transplantation (BMT) or...autologous bone marrow transplantation (ABMT) have produced encouraging results. In this report, we update our original study in which 20 patients with

  12. Current Status of Autologous Breast Tumor Cell-based Vaccines

    PubMed Central

    Kurtz, Samantha L.; Ravindranathan, Sruthi; Zaharoff, David A.

    2015-01-01

    Summary Approximately 9 of 10 breast cancer-related deaths are attributable to metastasis. Yet, less than 4% of breast cancer patients are initially diagnosed with metastatic cancer. Therefore, the majority of breast cancer-related deaths are due to recurrence and progression of nonmetastatic disease. There is tremendous clinical opportunity for novel adjuvant strategies, such as immunotherapies, that have the potential to prevent progressive recurrences. In particular, autologous tumor cell-based vaccines can train a patient's immune system to recognize and eliminate occult disease. Autologous tumor cell-based vaccines have several advantages including safety, multivalency and patient specificity. Furthermore, because lumpectomy or mastectomy is indicated for the vast majority of breast cancer patients, resected tumors offer a readily available, patient-specific source of tumor antigen. Disadvantages of autologous tumor cell-based vaccines include poor immunogenicity and production inconsistencies. This review summarizes recent progress in the development of autologous breast tumor vaccines and offers insight for overcoming existing limitations. PMID:25308888

  13. Canine Cranial Reconstruction Using Autologous Bone Marrow Stromal Cells

    PubMed Central

    Mankani, Mahesh H.; Kuznetsov, Sergei A.; Shannon, Brian; Nalla, Ravi K.; Ritchie, Robert O.; Qin, Yixian; Robey, Pamela Gehron

    2006-01-01

    Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects. PMID:16436668

  14. Lack of autologous tissue transmission of eosinophilic plaques in cats.

    PubMed

    Moriello, K A; Kunkle, G; Miller, L M; Crowley, A

    1990-07-01

    Autologous tissue transmission of spontaneously developing feline eosinophilic plaques was attempted in 5 cats. Macerated tissue from the plaque was vigorously rubbed onto 2 scarified skin sites in each cat. The inoculated areas were observed daily for 30 days. During that time, no clinical or histologic evidence of transmission was found.

  15. How Effective is Autologous Serum Therapy in Chronic Autoimmune Urticaria

    PubMed Central

    Majid, Imran; Shah, Shazia; Hassan, Altaf; Aleem, Saima; Aziz, Khalid

    2015-01-01

    Background: Chronic autoimmune urticaria (CAU) is one of the most challenging therapeutic problems faced by a dermatologist. Recently, weekly autologous serum injections have been shown to induce a prolonged remission in this disease. Aim: To evaluate the efficacy of repeated autologous serum injections in patients with CAU. Materials and Methods: Seventy patients of CAU were prospectively analyzed for the efficacy of nine consecutive weekly autologous serum injections with a post-intervention follow-up of 12 weeks. Total urticaria severity score (TSS) was monitored at the baseline, at the end of treatment and lastly at the end of 12 weeks of follow up. Response to treatment was judged by the percentage reduction in baseline TSS at the end of treatment and again at the end of 12 weeks-follow-up. Results: Out of the 70 patients enrolled, 11 dropped out of the injection treatment after one or the first few doses only. Among the rest of 59 patients, only 7 patients (12%) went into a partial or complete remission and remained so over the follow-up period of 12 weeks. Forty patients (68%) did not demonstrate any significant reduction in TSS at the end of the treatment period. Rest of the 12 patients showed either a good or excellent response while on weekly injection treatment, but all of them relapsed over the follow-up period of 12 weeks. Conclusion: Autologous serum therapy does not seem to lead to any prolonged remission in patients of CAU. PMID:25657418

  16. How Effective is Autologous Serum Therapy in Chronic Autoimmune Urticaria.

    PubMed

    Majid, Imran; Shah, Shazia; Hassan, Altaf; Aleem, Saima; Aziz, Khalid

    2015-01-01

    Chronic autoimmune urticaria (CAU) is one of the most challenging therapeutic problems faced by a dermatologist. Recently, weekly autologous serum injections have been shown to induce a prolonged remission in this disease. To evaluate the efficacy of repeated autologous serum injections in patients with CAU. Seventy patients of CAU were prospectively analyzed for the efficacy of nine consecutive weekly autologous serum injections with a post-intervention follow-up of 12 weeks. Total urticaria severity score (TSS) was monitored at the baseline, at the end of treatment and lastly at the end of 12 weeks of follow up. Response to treatment was judged by the percentage reduction in baseline TSS at the end of treatment and again at the end of 12 weeks-follow-up. Out of the 70 patients enrolled, 11 dropped out of the injection treatment after one or the first few doses only. Among the rest of 59 patients, only 7 patients (12%) went into a partial or complete remission and remained so over the follow-up period of 12 weeks. Forty patients (68%) did not demonstrate any significant reduction in TSS at the end of the treatment period. Rest of the 12 patients showed either a good or excellent response while on weekly injection treatment, but all of them relapsed over the follow-up period of 12 weeks. Autologous serum therapy does not seem to lead to any prolonged remission in patients of CAU.

  17. Detection of autologous blood transfusions in athletes: a historical perspective.

    PubMed

    Mørkeberg, Jakob

    2012-07-01

    Autologous blood transfusions (ABTs) has been used by athletes for approximately 4 decades to enhance their performance. Although the method was prohibited by the International Olympic Committee in the mid 1980s, no direct detection method has yet been developed and implemented by the World Anti-Doping Agency (WADA). Several indirect methods have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Compared with the first methods developed in 1987, the sensitivity of subsequent tests has not improved the detection of blood doping. Nevertheless, the use of sophisticated statistical algorithms has assured a higher level of specificity in subsequent detection models, which is a crucial aspect of antidoping testing particularly to avoid "false positives." Today, the testing markers with the best sensitivity/specificity ratio are the Hbmr model (an algorithm based on the total amount of circulating hemoglobin level [hemoglobin level mass] and percentage of reticulocytes, 4.51·ln(Hbmass)-√%ret) and the OFF-hr model (algorithm based on hemoglobin level concentration and percentage of reticulocytes, Hb(g/L)-60·√%ret). Only the OFF-hr model is currently approved by WADA. Recently, alternative indirect strategies for detecting blood doping have been proposed. One method is based upon a transfusion-induced immune-response resulting in specific changes in gene expression related to leukocytes such as T lymphocytes. Another method relies on detecting increased plasticizer metabolite levels in the urine caused by the leakage of plasticizers from the blood bags used during the blood storage. These methods need further development and validation across different types of transfusion regimes before they can be implemented. In addition, several research projects have been funded by WADA in recent years and are now under development including "Detection of Autologous Blood Transfusions Using Activated Red Blood Cells (the red blood cells

  18. Regeneration of Tissues and Organs Using Autologous Cells

    SciTech Connect

    Anthony Atala

    2010-04-28

    The Joint Commission for Health Care Organizations recently declared the shortage of transplantable organs and tissues a public health crisis. As such, there is about one death every 30 seconds due to organ failure. Complications and rejection are still significant albeit underappreciated problems. It is often overlooked that organ transplantation results in the patient being placed on an immune suppression regimen that will ultimate shorten their life span. Patients facing reconstruction often find that surgery is difficult or impossible due to the shortage of healthy autologous tissue. In many cases, autografting is a compromise between the condition and the cure that can result in substantial diminution of quality of life. The national cost of caring for persons who might benefit from engineered tissues or organs has reached $600 billion annually. Autologous tissue technologies have been developed as an alternative to transplantation or reconstructive surgery. Autologous tissues derived from the patient's own cells are capable of correcting numerous pathologies and injuries. The use of autologous cells eliminates the risks of rejection and immunological reactions, drastically reduces the time that patients must wait for lifesaving surgery, and negates the need for autologous tissue harvest, thereby eliminating the associated morbidities. In fact, the use of autologous tissues to create functional organs is one of the most important and groundbreaking steps ever taken in medicine. Although the basic premise of creating tissues in the laboratory has progressed dramatically, only a limited number of tissue developments have reached the patients to date. This is due, in part, to the several major technological challenges that require solutions. To that end, we have been in pursuit of more efficient ways to expand cells in vitro, methods to improve vascular support so that relevant volumes of engineered tissues can be grown, and constructs that can mimic the native

  19. Mode of formation and structural features of DNA-cationic liposome complexes used for transfection.

    PubMed

    Gershon, H; Ghirlando, R; Guttman, S B; Minsky, A

    1993-07-20

    Complexes formed between cationic liposomes and nucleic acids represent a highly efficient vehicle for delivery of DNA and RNA molecules into a large variety of eukaryotic cells. By using fluorescence, gel electrophoresis, and metal-shadowing electron microscopy techniques, the factors that affect the, yet unclear, interactions between DNA and cationic liposomes as well as the structural features of the resulting complexes have been elucidated. A model is suggested according to which cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, namely, DNA-induced membrane fusion, indicated by lipid mixing studies, and liposome-induced DNA collapse, pointed out by the marked cooperativity of the encapsulation processes, by their modulations by DNA-condensing agents, and also by their conspicuous independence upon DNA length. The DNA collapse leads to the formation of condensed structures which can be completely encapsulated within the fused lipid bilayers in a fast, highly cooperative process since their exposed surface is substantially smaller than that of extended DNA molecules. The formation of the transfecting DNA-liposome complexes in which the nucleic acids are fully encapsulated within a positively-charged lipid bilayer is proposed, consequently, to be dominated by mutual effects exerted by the DNA and the cationic liposomes, leading to interrelated lipid fusion and DNA collapse.

  20. Persistence of Virus Reservoirs in ART-Treated SHIV-Infected Rhesus Macaques after Autologous Hematopoietic Stem Cell Transplant

    PubMed Central

    Lawson, Benton; Lee, S. Thera; Chahroudi, Ann; Kean, Leslie; Silvestri, Guido

    2014-01-01

    Despite many advances in AIDS research, a cure for HIV infection remains elusive. Here, we performed autologous hematopoietic stem cell transplantation (HSCT) in three Simian/Human Immunodeficiency Virus (SHIV)-infected, antiretroviral therapy (ART)-treated rhesus macaques (RMs) using HSCs collected prior to infection and compared them to three SHIV-infected, ART-treated, untransplanted control animals to assess the effect of conditioning and autologous HSCT on viral persistence. As expected, ART drastically reduced virus replication, below 100 SHIV-RNA copies per ml of plasma in all animals. After several weeks on ART, experimental RMs received myeloablative total body irradiation (1080 cGy), which resulted in the depletion of 94–99% of circulating CD4+ T-cells, and low to undetectable SHIV-DNA levels in peripheral blood mononuclear cells. Following HSC infusion and successful engraftment, ART was interrupted (40–75 days post-transplant). Despite the observed dramatic reduction of the peripheral blood viral reservoir, rapid rebound of plasma viremia was observed in two out of three transplanted RMs. In the third transplanted animal, plasma SHIV-RNA and SHIV DNA in bulk PBMCs remained undetectable at week two post-ART interruption. No further time-points could be assessed as this animal was euthanized for clinical reasons; however, SHIV-DNA could be detected in this animal at necropsy in sorted circulating CD4+ T-cells, spleen and lymph nodes but not in the gastro-intestinal tract or tonsils. Furthermore, SIV DNA levels post-ART interruption were equivalent in several tissues in transplanted and control animals. While persistence of virus reservoir was observed despite myeloablation and HSCT in the setting of short term ART, this experiment demonstrates that autologous HSCT can be successfully performed in SIV-infected ART-treated RMs offering a new experimental in vivo platform to test innovative interventions aimed at curing HIV infection in humans. PMID

  1. Mechanical and chemical characteristics of an autologous glue.

    PubMed

    De Somer, Filip; Delanghe, Joris; Somers, Pamela; Debrouwere, Maarten; Van Nooten, Guido

    2008-09-15

    The study evaluates the mechanical and chemical characteristics of autologous surgical glue made by mixing ultrafiltered plasma with glutaraldehyde (GTA). Human albumin 200 g/L mixed with different concentrations of GTA (25, 50, 75, or 100 g/L) was used as a single protein set-up for testing tensile strength, elasticity, and rate of crosslinking. Subsequently, ultrafiltered canine or human plasma to obtain autologous glue replaced human albumin. BioGlue, a surgical glue, and Tissucol Duo, a fibrin sealant, were used as controls. Tensile strength of human albumin 200 g/L mixed with 75 g/L GTA is 825 +/- 109 N versus 672 +/- 167 N for BioGlue. Ultrafiltered canine plasma showed a maximum tensile strength of 634 +/- 137 N when mixed with GTA 75 g/L. For human plasma, the maximum tensile strength of 436 +/- 69 N was reached after mixing with GTA 25 g/L. Autologous glue had a higher elasticity of 144 +/- 66 N versus 322 +/- 104 N for BioGlue at maximum load. Autologous glues for vascular repair can be easily prepared out of the patient's plasma. The optimal characteristics, compared to BioGlue, are obtained for ultrafiltered canine and human plasma by mixing with a GTA concentration of 50-75 g/L and 25-50 g/L, respectively. The autologous glue will exert less tensile strength than BioGlue but has a better compliance. In case where no plasma can obtained from the patient, mixing human albumin 200 g/L with GTA 75 g/L can be an alternative to BioGlue.

  2. Use of containers with sterilizing filter in autologous serum eyedrops.

    PubMed

    López-García, José S; García-Lozano, Isabel

    2012-11-01

    To assess the effect of the use of containers with an adapted sterilizing filter on the contamination of autologous serum eyedrops. Prospective, consecutive, comparative, and randomized study. Thirty patients with Sjögren syndrome. One hundred seventy-six autologous serum containers used in home therapy were studied; 48 of them included an adapted filter (Hyabak; Thea, Clermont-Ferrand, France), and the other 128 were conventional containers. Containers equipped with a filter were tested at 7, 14, 21, and 28 days of use, whereas conventional containers were studied after 7 days of use. In addition, testing for contamination was carried out in 14 conventional containers used during in-patient therapy every week for 4 weeks. In all cases, the preparation of the autologous serum was similar. Blood agar and chocolate agar were used as regular culture media for the microbiologic studies, whereas Sabouraud agar with chloramphenicol was the medium for fungal studies. Microbiologic contamination of containers with autologous serum eyedrops. Only one of the containers with an adapted sterilizing filter (2.1%) became contaminated with Staphylococcus epidermidis after 1 month of treatment, whereas the contamination rate among conventional containers reached 28.9% after 7 days of treatment. The most frequent germs found in the samples were coagulase-negative Staphylococcus (48.6%). With regard the containers used in the in-patient setting, 2 (14.3%) became contaminated after 2 weeks, 5 (35.7%) became contaminated after 3 weeks, and 5 (50%) became contaminated after 4 weeks, leaving 7 (50%) that did not become contaminated after 1 month of treatment. Using containers with an adapted filter significantly reduces the contamination rates in autologous serum eyedrops, thus extending the use of such container by the patients for up to 4 weeks with virtually no contamination risks. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  3. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

    PubMed

    Ferizi, Mehrije; Aneja, Manish K; Balmayor, Elizabeth R; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-12-15

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

  4. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

    PubMed Central

    Ferizi, Mehrije; Aneja, Manish K.; Balmayor, Elizabeth R.; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-01-01

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs. PMID:27974853

  5. 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

  6. Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

    PubMed

    Mialhe, E; Miller, L H

    1994-05-01

    To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

  7. Software-aided automatic laser optoporation and transfection of cells

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

    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.

  8. Transfection of human prostate cancer CA-HPV-10 cells with cytosolic sulfotransferase SULT1E1 affects estrogen signaling and gene transcription.

    PubMed

    Kapoor, Ruchita; Sheng, Jonathan J

    2008-02-01

    Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of estrogens and estrogenic drugs in human reproductive tissues. Logically, this estrogen-preferring sulfotransferase isoform could play a regulatory role in estrogen signaling activities in human reproductive cells, including the prostate cells. This hypothesis was tested using DNA microarray and real-time reverse transcription-polymerase chain reaction methods in the present work. Potential changes in the transcriptional expression of selected signal transduction-related genes in human prostate cancer CA-HPV-10 cell line after SULT1E1 transfection were examined by DNA microarray methods. Notable changes were observed in the mRNA expression levels of TFRC, a cell membrane transferrin receptor gene, and TMEPAI, a gene encoding a steroid-dependent mRNA product. Expression of TFRC was down-regulated, whereas expression of TMEPAI was up-regulated by SULT1E1 transfection in CA-HPV-10 cells. Data from the current studies also showed that the estrogen-induced estrogen response element activation in CA-HPV-10 cells was repressed after the cells were transfected with SULT1E1. These results indicate that SULT1E1 may function as a transcriptional mediator in human prostate cancer CA-HPV-10 cells.

  9. Design, synthesis, and transfection biology of novel cationic glycolipids for use in liposomal gene delivery.

    PubMed

    Banerjee, R; Mahidhar, Y V; Chaudhuri, A; Gopal, V; Rao, N M

    2001-11-22

    The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

  10. Effects of Lipofectamine 2000/siRNA complexes on autophagy in hepatoma cells.

    PubMed

    Mo, Robert H; Zaro, Jennica L; Ou, Jing-Hsiung James; Shen, Wei-Chiang

    2012-05-01

    Lipofectamine 2000 is commonly used for siRNA transfections. However, few studies have examined cellular responses to this delivery system. The purpose of this study is to evaluate the effect of siRNA transfection using Lipofectamine 2000 on cellular autophagy. Huh7.5 cells, stably transfected to express GFP-LC3, were treated with Lipofectamine 2000/negative control siRNA (NC siRNA) complexes. At different time points after treatment, cells were lysed and analyzed by immunoblotting and fluorescence spectroscopy. Cells were also observed using confocal microscopy. An increase of endogenous LC3 lipidation, GFP-LC3 fluorescence, and autophagosomal puncta was observed in cells treated with Lipofectamine 2000/NC siRNA complexes. The kinetics of the increase of GFP-LC3 fluorescence correlated with the concentration of NC siRNA transfected, where 50, 100, and 200 nM NC siRNA caused a significant increase at 72, 48, and 24 h, respectively, after transfection. A similar effect on the GFP-LC3 signal was also observed for cells treated with Lipofectamine 2000 complexed with two other NC siRNAs. The effects were also confirmed in another hepatoma cell line, H4IIE, by immunoblotting. Lipofectamine 2000-mediated transport of NC siRNAs led to an increase of autophagosomes in a dose- and time-dependent manner. Thus, this effect on cells should be taken into consideration when using this approach for intracellular delivery of siRNA.

  11. MicroRNA-27b Enhances the Hepatic Regenerative Properties of Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Chen, Kuang-Den; Huang, Kuang-Tzu; Lin, Chih-Che; Weng, Wei-Teng; Hsu, Li-Wen; Goto, Shigeru; Nakano, Toshiaki; Lai, Chia-Yun; Kung, Chao-Pin; Chiu, King-Wah; Wang, Chih-Chi; Cheng, Yu-Fan; Ma, Yen-Ying; Chen, Chao-Long

    2016-01-01

    Adipose-derived mesenchymal stem cells (ASCs) are readily available multipotent mesenchymal progenitor cells and have become an attractive therapeutic tool for regenerative medicine. We herein investigated the mechanistic role of how miR-27b modulated regenerative capacities of ASCs. Intravenous administration of miR-27b-transfected ASCs (ASCs-miR-27b) was conducted after 70% partial hepatectomy (PH). After PH, rats injected with ASCs-miR-27b had decreased inflammatory cytokines and increased hepatocyte growth factor and other related growth factors. We showed that the nature of ASCs-miR-27b to inhibit hepatic stellate cell activation was dependent upon peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in vitro. Moreover, expression of miR-27b in ASCs induced heme oxygenase-1 (HO-1), resulting in increased production of ATP, protective cytokines/growth factors, and genes involved in mitochondrial biogenesis in a PGC-1α-dependent manner. RNA sequencing (RNA-Seq) analysis revealed drastic transcriptional changes in livers treated with ASCs-miR-27b after PH. The differentially expressed genes classified into “regeneration,” “fibrosis,” and “mitochondrial biogenesis” clusters were mainly mitochondrial. The potential biological context reflecting the effects of PGC-1α by ASCs-miR-27b treatment was also observed by the subnetwork analysis with HO-1 and PGC-1α being the top-ranked regulatory genes. We demonstrate autologous ASCs-miR-27b enhances liver regeneration and, importantly, preserves hepatic function through paracrine actions which offers a viable therapeutic option to facilitate rapid recovery after liver injury. PMID:26836372

  12. hTERT mRNA dendritic cell vaccination: complete response in a pancreatic cancer patient associated with response against several hTERT epitopes.

    PubMed

    Suso, Else M Inderberg; Dueland, Svein; Rasmussen, Anne-Marie; Vetrhus, Turid; Aamdal, Steinar; Kvalheim, Gunnar; Gaudernack, Gustav

    2011-06-01

    Immunotherapy targeting the hTERT subunit of telomerase has been shown to induce robust immune responses in cancer patients after vaccination with single hTERT peptides. Vaccination with dendritic cells (DCs) transfected with hTERT mRNA has the potential to induce strong immune responses to multiple hTERT epitopes and is therefore an attractive approach to more potent immunotherapy. Blood samples from such patients provide an opportunity for identification of new, in vivo processed T-cell epitopes that may be clinically relevant. A 62-year-old female patient underwent radical surgery for a pancreatic adenocarcinoma. After relapse, she obtained stable disease on gemcitabine treatment. Due to severe neutropenia, the chemotherapy was terminated. The patient has subsequently been treated with autologous DCs loaded with hTERT mRNA for 3 years. Immunomonitoring was performed at regular intervals following start of vaccination and clinical outcome measured by CT and PET/CT evaluation. The patient developed an immune response against several hTERT-derived Th and CTL epitopes. She presently shows no evidence of active disease based on PET/CT scans. No serious adverse events were experienced and the patient continues to receive regular booster injections. We here provide evidence for the induction of hTERT-specific immune responses following vaccination of a pancreas cancer patient with DCs loaded with hTERT mRNA. These responses are associated with complete remission. A thorough analysis of this patient immune response has provided a unique opportunity to identify novel epitopes, associated with clinical effects. These will be included in future hTERT vaccines.

  13. Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma

    PubMed Central

    Mazzocco, Marta; Martini, Matteo; Rosato, Antonio; Stefani, Elisabetta; Matucci, Andrea; Dalla Santa, Silvia; De Sanctis, Francesco; Ugel, Stefano; Sandri, Sara; Ferrarini, Giovanna; Cestari, Tiziana; Ferrari, Sergio; Zanovello, Paola; Bronte, Vincenzo; Sartoris, Silvia

    2015-01-01

    In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL) -mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 Ld. Increase of H-2 Ld expression by cDNA transfection (Sp6/B7/Ld) raised tumour immune protection and shifted most CTL responses towards H-2 Ld-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 Ld-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/Ld cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost. PMID:25959091

  14. Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma.

    PubMed

    Mazzocco, Marta; Martini, Matteo; Rosato, Antonio; Stefani, Elisabetta; Matucci, Andrea; Dalla Santa, Silvia; De Sanctis, Francesco; Ugel, Stefano; Sandri, Sara; Ferrarini, Giovanna; Cestari, Tiziana; Ferrari, Sergio; Zanovello, Paola; Bronte, Vincenzo; Sartoris, Silvia

    2015-09-01

    In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL)-mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 L(d). Increase of H-2 L(d) expression by cDNA transfection (Sp6/B7/L(d)) raised tumour immune protection and shifted most CTL responses towards H-2 L(d)-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 L(d)-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/L(d) cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

  15. RNA genetics

    SciTech Connect

    Domingo, E. ); Holland, J.J. . Dept. of Biology); Ahlquist, P. . Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: Retroviruses, Viroids, and RNA recombination, Volume 2. Topics covered include: Replication of retrovirus genomes, Hepatitis B virus replication, and Evolution of RNA viruses.

  16. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    PubMed

    Liu, Lin-Lin; Zhao, Hui; Ma, Teng-Fei; Ge, Fei; Chen, Ce-Shi; Zhang, Ya-Ping

    2015-01-01

    Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

  17. [Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2].

    PubMed

    Chen, Hui; Zhu, Heng; Chu, Ya-Nan; Xu, Fen-Fen; Liu, Yuan-Lin; Tang, Bo; Li, Xi-Mei; Hu, Liang-Ding; Zhang, Yi

    2014-10-01

    This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.

  18. Increased sensitivity to Vinca alkaloids in cells overexpressing calmodulin by gene transfection.

    PubMed

    Ido, M; Lagacé, L; Chafouleas, J G

    1990-10-15

    Mouse C127 cells, transfected with the chicken calmodulin (CaM) gene and overexpressing CaM protein, were used to evaluate the effect of elevated levels of CaM on the sensitivity of these cells to various anticancer drugs. Clones C2 and C3 overexpress CaM mRNA by 40- and 80-fold, respectively, and CaM protein 3- and 8-fold, respectively. These cell lines were tested for their sensitivity to vincristine, vinblastine, bleomycin, and Adriamycin by comparing the 50% inhibitory concentration in a 72-h growth inhibition assay. The 50% inhibitory concentration values for vincristine with C2 and C3 cells were 6.27 +/- 0.56 nM and 6.60 +/- 0.96 nM, respectively. These values were significantly lower than 13.9 +/- 0.79 nM for the parental C127 cells and 14.0 +/- 1.55 nM for clone 6.8 (the control cell line for transfection without the chicken CaM gene) at P less than or equal to 0.005. The proliferation of C2 and C3 cells was inhibited at lower concentrations of vinblastine as well. The 50% inhibitory concentration values for the C2 and C3 cell lines were approximately one-half those required for C127 or clone 6.8 cells. However, no significant difference in the sensitivity to the DNA-binding drugs, bleomycin and Adriamycin, was observed between the different cell lines. The uptake of [3H]vinblastine was evaluated and found to be increased 1.6- and 2.8-fold in C2 and C3 cells, respectively, as compared with that value obtained for C127 cells. Moreover, the efflux of [3H]vinblastine from vinblastine-loaded cells was also observed to be decreased in the C2 and C3 cell lines. These data suggest that the increase in CaM expression in the C2 and C3 cell lines might be related to the higher sensitivity of these cells to Vinca alkaloids. This increased sensitivity appears to be due to the increase in intracellular concentration of the Vinca alkaloids as a result of an increase in drug uptake and a decrease in efflux. Moreover, the increased sensitivity of clones C2 and C3 to Vinca

  19. Targeted surface expression of an exogenous antigen in stably transfected babesia bovis

    USDA-ARS?s Scientific Manuscript database

    Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected ...

  20. Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

    PubMed

    Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

    2016-10-01

    High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Phospholipid-modified polyethylenimine-based nanopreparations for siRNA–mediated gene silencing: Implications for transfection and the role of lipid components

    PubMed Central

    Navarro, Gemma; Essex, Sean; Sawant, Rupa R.; Biswas, Swati; Nagesha, Dattatri; Sridhar, Srinivas; de ILarduya, Conchita Tros; Torchilin, Vladimir P.

    2013-01-01

    The clinical application of gene silencing mediated by small interfering RNA (siRNA) has been limited by the lack of efficient and safe carriers. Phospholipid modification of low molecular weight polyethylenimine (PEI 1.8 kDa) dramatically increased its gene down-regulation capacity while keeping cytotoxicity levels low. The silencing efficacy was highly dependent on the nature of the lipid grafted to PEI and the polymer/siRNA ratio employed. Phosphoethanolamine (DOPE and DPPE) and phosphocholine (PC) conjugation did not change the physicochemical properties and siRNA binding capacity of PEI complexes but had a large impact on their transfection and ability to downregulate Green Fluorescent Protein (GFP) expression (60%, 30% and 5% decrease of GFP expression respectively). We found that the micelle-forming structure of DOPE and DPPE-PEI dramatically changed PEI’s interaction with cell membranes and played a key role in promoting PEI 1.8 kDa transfection, completely ineffective in the absence of the lipid modification. PMID:23928214

  2. The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

    PubMed Central

    Stalder, Lukas; Heusermann, Wolf; Sokol, Lena; Trojer, Dominic; Wirz, Joel; Hean, Justin; Fritzsche, Anja; Aeschimann, Florian; Pfanzagl, Vera; Basselet, Pascal; Weiler, Jan; Hintersteiner, Martin; Morrissey, David V; Meisner-Kober, Nicole C

    2013-01-01

    Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing. PMID:23511973

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

    PubMed Central

    2017-01-01

    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

  4. 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

  5. Regenerative effect of hOPG gene-modified autologous PDLs in combination with cell transplantation on periodontal defection in beagle dogs.

    PubMed

    Jiang, Su; Tang, Kunqi; Chen, Bin; Yan, Fuhua

    2016-12-01

    This study evaluated the ability of human osteoprotegerin gene-modified autologous periodontal ligament cells (PDLCs) in combination with cell transplantation to promote periodontal regeneration in beagle dogs. Adenovirus Ad5-hOPG-EGFP-transfected PDLCs and BME-10X collagen membranes were fabricated and used for periodontal repair. Buccal periodontal defects (mesiodistal width × depth: 5 × 5 mm) were created on the second, third, and fourth mandibular premolars in six normal beagle dogs, and the defects were histologically and histomorphometrically assessed for periodontal regeneration in the following four groups: (1) hOPG-PDLCs + BME-10X, (2) mock-PDLCs + BME-10X, (3) PDLCs + BME-10X, and (4) BME-10X. The radiographic and histological results suggested that hOPG-PDLCs significantly promoted periodontal defect repair. This study demonstrates the potential of hOPG-modified PDLCs for periodontal tissue regeneration.

  6. Gene printer: laser-scanning targeted transfection of cultured cardiac neonatal rat cells.

    PubMed

    Nikolskaya, Alena V; Nikolski, Vladimir P; Efimov, Igor R

    2006-01-01

    Heterogeneous gene expression in cardiac cells and tissues which requires targeted delivery of foreign DNA into selected cells or regions is needed for the development of novel therapies. Several techniques have been employed for targeted transfection, such as direct microinjection into cells or targeted electroporation. However, these techniques have limited bandwidth or spatial resolution of transfection. We aimed to develop a method for transfection of cardiac cells by means of laser-assisted optoporation using a standard confocal microscope. This technique allows for the transfection of selected cell types in the presence of other cell types as long as they are distinguishable with a microscope. This technique can work as a "gene printer" creating arbitrarily shaped areas of transfected cells.

  7. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    SciTech Connect

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  8. Transfection of eggs in the bivalve mollusc Chamelea gallina (Bivalvia, Veneridae).

    PubMed

    Guerra, R; Esponda, P

    2006-04-01

    Eggs from the bivalve mollusc Chamelea gallina were transfected in vitro. The p-GeneGrip gene construction that expresses the green fluorescent protein (GFP) was employed. It was necessary to remove the jelly coat which covers the egg surface for a successful transfection, and then 44.2% of gametes appeared transfected after using naked DNA. On the other hand, cationic liposomes (Lipofectamine) and neutral lipids (GenePORTER) were employed as gene vectors. After the employ of Lipofectamine 35.6% of eggs were transfected and 41.4% after using GenePORTER. Fluorescence analysis showed that the foreign gene appeared principally located in the egg cytoplasm, but laser confocal microscopy showed that it was also present in the nucleus. Furthermore, PCR analysis demonstrated that the foreign DNA appeared in the DNA extracted from the treated eggs. This simple method for the transfection of mollusc eggs would be interesting for future biotechnological applications in species of commercial interest.

  9. 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.

  10. Contrasting effects of cysteine modification on the transfection efficiency of amphipathic peptides.

    PubMed

    Sharma, Rajpal; Nisakar, Daniel; Shivpuri, Shivangi; Ganguli, Munia

    2014-08-01

    Delivery of DNA to cells remains a key challenge towards development of gene therapy. A better understanding of the properties involved in stability and transfection efficiency of the vector could critically contribute to the improvement of delivery vehicles. In the present work we have chosen two peptides differing only in amphipathicity and explored how presence of cysteine affects DNA uptake and transfection efficiency. We report an unusual observation that addition of cysteine selectively increases transfection efficiency of secondary amphipathic peptide (Mgpe-9) and causes a drop in the primary amphipathic peptide (Mgpe-10). Our results point the effect of cysteine is dictated by the importance of physicochemical properties of the carrier peptide. We also report a DNA delivery agent Mgpe-9 exhibiting high transfection efficiency in multiple cell lines (including hard-to-transfect cell lines) with minimal cytotoxicity which can be further explored for in vivo applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

    PubMed

    Haines, A M; Irvine, A S; Mountain, A; Charlesworth, J; Farrow, N A; Husain, R D; Hyde, H; Ketteringham, H; McDermott, R H; Mulcahy, A F; Mustoe, T L; Reid, S C; Rouquette, M; Shaw, J C; Thatcher, D R; Welsh, J H; Williams, D E; Zauner, W; Phillips, R O

    2001-01-01

    Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

  12. Transfection with replicating DNA from the temperate Bacillus bacteriophage phi 105 and with T4-ligase treated phi105 DNA: the importance in transfection of being longer than genome-length.

    PubMed

    Flock, J I

    1978-07-06

    Replicating phage DNA extracted from Bacillus subtilis infected with phage phi 105 has a higher activity in transfection than mature DNA. By heteroduplex analysis it was shown that this DNA contains concatemeric molecules. Concatemers, constructed in vitro by treatment of mature DNA with T4-ligase also have an increased activity in transfection. DNA showing an increased activity in transfection does not have a requirement for more than one molecule per transfection event as is typically found for transfection with mature phi 105 DNA. An explanation is given for this difference suggesting that the structure of the ends of the transfecting molecules play an important role intransfection.

  13. Processing of proinsulin by transfected hepatoma (FAO) cells.

    PubMed

    Vollenweider, F; Irminger, J C; Gross, D J; Villa-Komaroff, L; Halban, P A

    1992-07-25

    Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.

  14. Efficient non-viral gene delivery mediated by nanostructured calcium carbonate in solution-based transfection and solid-phase transfection.

    PubMed

    Chen, Si; Li, Feng; Zhuo, Ren-Xi; Cheng, Si-Xue

    2011-10-01

    Among different non-viral gene delivery methods, the technique of co-precipitation of Ca(2+) with DNA in the presence of inorganic anions is an attractive option because of the biocompatibility and biodegradability. In this study, nano-sized CaCO(3)/DNA co-precipitates for gene delivery were prepared. The effect of Ca(2+)/CO(3)(2-) molar ratio on the gene delivery was investigated. The mechanism of the transfection mediated by CaCO(3)/DNA co-precipitates was studied by treatment of the cells with chloroquine, wortmannin and cytochalasin D, respectively. The in vitro gene transfections in different cells were carried out for both solution-based transfection and solid-phase transfection. The gene expression of the calcium carbonate based approach is strongly affected by the Ca(2+)/CO(3)(2-) ratio because the size of CaCO(3)/DNA co-precipitates is mainly determined by the Ca(2+)/CO(3)(2-) ratio. In addition, the encapsulation efficiency of DNA increases with decreasing Ca(2+)/CO(3)(2-) ratio. With a suitable Ca(2+)/CO(3)(2-) ratio, CaCO(3)/DNA co-precipitates could effectively mediate gene transfection with the expression levels higher than that of Lipofectamine 2000 in the presence of serum. The mechanism study shows that CaCO(3)/DNA co-precipitates are internalized via endocytosis of the cells and macropinocytosis is the main route of internalization. Compared with the solution-based transfection, CaCO(3)/DNA co-precipitates in the solid-phase transfection exhibit a lower gene expression level. The calcium carbonate based approach has great potential in gene delivery.

  15. Chitosan/poly(vinyl alcohol) hydrogel combined with Ad-hTGF-β1 transfected mesenchymal stem cells to repair rabbit articular cartilage defects.

    PubMed

    Qi, Bai-wen; Yu, Ai-xi; Zhu, Shao-bo; Zhou, Min; Wu, Gang

    2013-01-01

    The aim of this work is to explore the feasibility and therapeutic effect of repairing rabbit articular cartilage defects using thermo-sensitive chitosan/poly (vinyl alcohol) composite hydrogel engineered Ad-hTGF-β1-transfected bone marrow mesenchymal stem cells. Rabbit's bone marrow stromal cells (BMSCs) were obtained and cultured in vitro and transfected with a well-constructed Ad-hTGF-β1 vector, the cartilage phenotype of the transfected cells was tested by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Twenty-four New Zealand white rabbits with articular cartilage defects were randomly divided into four groups: group A was treated with CS/PVA gel and transfected BMSCs; group B received CS/PVA gel and un-transfected BMSCs; group C was treated with CS/PVA gel alone and group D was the untreated control group. Experimental animals of each group were killed at 16 weeks after operation. General observation, Masson's trichrome staining and collagen II immunohistological staining of the specimens were performed to evaluate the repair effect. The Wakitani scoring method was used to evaluate the repair effect. RT-PCR and Western blot confirmed that the hTGF-β1 gene was expressed in BMSCs and triggered the expression of specific markers of cartilage differentiation such as aggrecan mRNA and Collagen II in BMSCs after transfection with Ad-hTGF-β1. Sixteen weeks after operation, the defects in group A had smooth and flat surfaces, and the defects appeared to have completely healed, exhibiting almost the same color and texture as the surrounding cartilage. Masson's trichrome staining showed that the cell arrangement and density of regenerated cartilage tissue in group A was not significantly different from that of normal cartilage tissue. The immunohistochemical staining of Col II showed a strong expression in group A and weak expression in group B, but no expression in groups C and D. According to the Wakitani score, the difference between

  16. [Application of autologous fat grafting in breast reconstruction].

    PubMed

    Cai, L; Han, X F; Wang, B Q; Li, F C

    2017-09-01

    Objective: To observe the outcome of breast reconstruction with autologous fat grafting in the patients following treatment for breast cancer. Methods: The clinical data of 22 patients after breast cancer modified radical mastectomy with fat grafting for breast reconstruction from January 2012 to March 2015 at Department of Body Contouring and Liposuction Center of Plastic Surgery, Hospital of Peking Union Medical College were analyzed retrospectively. The age of 22 patients (all female) was 28 to 54 years. Fifteen patients were performed breast modified radical mastectomy 5 to 16 year ago without radiotherapy, 7 patients were performed breast modified radical mastectomy following regular radiotherapy 2 years ago. Low negative pressure liposuction technical was applied to harvest fat tissue for 400 to 800 ml which was filtrated and purified by cotton pad method in low temperature environment. Fat grafting was performed with multi-level and multi-tunnel and in multi-point injection ways. All patients were followed up by regular imaging evaluation with MRI or ultrasonography after operation every 3 months. Results: All breast reconstruction were successfully performed in 22 patients, no severe complications occurred. Among 15 patients without radiotherapy, 12 patients were performed with autologous fat grafting for breast reconstruction, 3 patients with prosthetic implantation for breast augmentation after autologous fat grafting. Among 7 patients with radiotherapy, 6 patients were performed with autologous fat grafting for breast reconstruction, 1 patient with prosthetic implantation for breast augmentation after autologous fat grafting. The volume of fat grafting was 104 to 380 ml. It took 2.5 hours to finish the operation including 1.0 to 1.5 hours for liposuction and 40 minutes for fat grafting. Next fat grafting were performed after 3 months. The fat of the breast were survived well detecting by MRI, only 1 patient had a cystic nodule which had been resected

  17. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  18. Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields.

    PubMed

    Xie, T D; Tsong, T Y

    1990-10-01

    Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field. Transfection efficiency varied with the waveform: square wave > sine wave > triangle wave. If the DNA was added after the ac field was turned off, transfection efficiency was reduced to the background level within 1 min. The field intensity used in this study was low and insufficient to cause electric breakdown of cell membranes. Thus, DNA

  19. MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

    PubMed Central

    Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

    2014-01-01

    Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

  20. A three-dimensional tumor cell defect in activating autologous CTLs is associated with inefficient antigen presentation correlated with heat shock protein-70 down-regulation.

    PubMed

    Dangles-Marie, Virginie; Richon, Sophie; El-Behi, Mohamed; Echchakir, Hamid; Dorothée, Guillaume; Thiery, Jérôme; Validire, Pierre; Vergnon, Isabelle; Menez, Jeanne; Ladjimi, Moncef; Chouaib, Salem; Bellet, Dominique; Mami-Chouaib, Fathia

    2003-07-01

    We described previously a CTL clone able to lyse the autologous carcinoma cell line IGR-Heu after specific recognition of an HLA-A2/mutated alpha-actinin-4 peptide complex. Here, we used IGR-Heu, cultured either as standard two-dimensional monolayers or as three-dimensional spheroids, to further analyze the influence of target architecture on CTL reactivity. Interestingly, we found that changes in the tumor structure from two- to three-dimensional induced a dramatic decrease in its capacity to activate autologous CTL, as measured by IFN-gamma and tumor necrosis factor-alpha secretion. These functional alterations were attributable neither to MHC class I expression nor to tumor antigen (Ag) down-regulation, because IGR-Heu, cultured as two- or three-dimensional, expressed similar levels of HLA-A2 and alpha-actinin-4. More importantly, incubation of three-dimensional cells with synthetic epitope completely restored cytokine release by CTL. This defective Ag presentation correlated with a decrease in heat shock protein (hsp)70 expression by three-dimensional tumors compared with two-dimensional cells. Furthermore, transfection of the tumor cells with hsp70 cDNA completely restored the Ag-presenting potential of spheroids and, therefore, cytokine production by T cells. These data strongly suggest that hsp70 down-regulation in three-dimensional cells may result in tumor resistance to the immune response.

  1. Autologous blood clot embolisation in posttraumatic high-flow priapism.

    PubMed

    Akpinar, Suha; Yilmaz, Güliz

    2016-11-01

    Non-ischemic, high-flow priapism is defined as the state of painless and permanent erection of the penis which generally develops by perineal trauma. Selective transarterial embolisation is one of the treatment options. We present an 18-year-old men who had complaints of painless and permanent erection after a blunt perineal trauma. Colour Doppler ultrasound revealed a pseudoaneurysm and fistula at the left cavernosal artery. Hence autologous blood clot injection was performed to embolise the pseudoaneurysm. Due to the recanalization on the postprocedural seventh day, second embolisation was performed. One month after the second procedure, colour Doppler ultrasound revealed a 50% shrink but mild refilling in the pseudoaneurysm, whereas complete thrombus formation was observed on follow-up imaging. His priapism had fully recovered and erectile functions were totally normal at the six months and one year follow up. Autologous blood clot embolisation seems as a safe and successful treatment. © The Author(s) 2015.

  2. Defective autologous mixed lymphocyte reactivity in multiple sclerosis.

    PubMed Central

    Hirsch, R L

    1986-01-01

    T cells from patients with multiple sclerosis (MS) and normal controls were assessed for their ability to respond in the autologous mixed lymphocyte reaction (AMLR). Cells from stable MS patients demonstrated a significant defect in their proliferative response to non-T cells in comparison to normal controls. Despite the defective AMLR response, T cells from MS patients reacted as well as T cells from normal controls to allogeneic stimuli. Furthermore, MS non-T-cells were fully capable of stimulating allogeneic MLR responses by normal and MS T cells. Since the T4+ cell is the major subpopulation which proliferates in the AMLR, these studies suggest a functional defect in a subpopulation of T4+ cells in MS patients. Since the AMLR may represent an important mechanism by which immune responses are regulated, a defect in the ability of MS T cells to respond to autologous cells could account for several of the autoimmune features of the disease. PMID:2942317

  3. SECOND AUTOLOGOUS STEM CELL TRANSPLANTATION FOR RELAPSED LYMPHOMA AFTER A PRIOR AUTOLOGOUS TRANSPLANT

    PubMed Central

    Smith, Sonali M.; van Besien, Koen; Carreras, Jeanette; Bashey, Asad; Cairo, Mitchell S.; Freytes, Cesar O.; Gale, Robert Peter; Hale, Gregory A.; Hayes-Lattin, Brandon; Holmberg, Leona A.; Keating, Armand; Maziarz, Richard T.; McCarthy, Philip L.; Navarro, Willis H.; Pavlovsky, Santiago; Schouten, Harry C.; Seftel, Matthew; Wiernik, Peter H.; Vose, Julie M.; Lazarus, Hillard M.; Hari, Parameswaran

    2012-01-01

    We determined treatment-related mortality (TRM), progression free survival (PFS), and overall survival (OS) after a second autologous HCT (HCT2) for patients with lymphoma relapse after a prior HCT (HCT1). Outcomes for patients with either Hodgkin lymphoma (HL, n=21) or non-Hodgkin lymphoma (NHL, n=19) receiving HCT2 reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) were analyzed. The median age at HCT2 was 38 years (range, 16–61) and 22 (58%) patients had a Karnofsky performance score less than 90. HCT2 was performed >1 year after HCT1 in 82%. The probability of TRM at day 100 was 15% (95% CI, 3–22%). The 1, 3 and 5 yr probabilities of PFS were 50% (95% CI, 34–66%), 36% (95% CI, 21–52%) and 30% (95% CI, 16–46%), respectively. Corresponding probabilities of survival were 65% (95% CI, 50–79%), 36% (95% CI, 22–52%) and 30% (95% CI, 17–46%), respectively. At a median follow up of 72 months (range, 12–124 months) after HCT2, 29 patients (73%) have died, 18 (62%) secondary to relapsed lymphoma. The outcomes of patients with HL and NHL were similar. In summary, this series represents the largest reported group of patients with relapsed lymphomas undergoing SCT2 following failed SCT1, and with long-term follow-up. Our series suggests that SCT2 is feasible in patients relapsing after prior HCT1, with a lower TRM than that reported for allogeneic transplant in this setting. HCT2 should be considered for patients with relapsed HL or NHL after HCT1 without alternative allogeneic stem cell transplant options. PMID:18640574

  4. Omnipotent RNA.

    PubMed

    Spirin, Alexander S

    2002-10-23

    The capability of polyribonucleotide chains to form unique, compactly folded structures is considered the basis for diverse non-genetic functions of RNA, including the function of recognition of various ligands and the catalytic function. Together with well-known genetic functions of RNA - coding and complementary replication - this has led to the concept of the functional omnipotence of RNA and the hypothesis that an ancient RNA world supposedly preceded the contemporary DNA-RNA-protein life. It is proposed that the Woese universal precursor in the ancient RNA world could be a cell-free community of mixed RNA colonies growing and multiplying on solid surfaces.

  5. Autologous Fat Transfer in a Patient with Lupus Erythematosus Profundus

    PubMed Central

    Yoon, Jimi; Kim, Hwa Mi; Kim, Tae-Heung; Kim, Chung-Won; Sun, Young-Woo; Yoon, Tae-Jin

    2012-01-01

    Lupus erythematosus profundus, a form of chronic cutaneous lupus erythematosus, is a rare inflammatory disease involving in the lower dermis and subcutaneous tissues. It primarily affects the head, proximal upper arms, trunk, thighs, and presents as firm nodules, 1 to 3 cm in diameter. The overlying skin often becomes attached to the subcutaneous nodules and is drawn inward to produce deep, saucerized depressions. We present a rare case of lupus erythematosus profundus treated with autologous fat transfer. PMID:23139658

  6. [History of autologous blood transfusion in neurosurgical operations].

    PubMed

    Nagai, M

    1998-12-01

    The first report on predeposit autologous blood transfusion was made in 1921 by F.C. Grant, neurosurgeon in the University Hospital of Pennsylvania. The patient was a 42-year-old man with cerebellar tumor, having a rare blood type for which no donor had been listed up. 500 ml of autologous blood was obtained, kept in 0.2% sodium citrate solution in a refrigerator and retransfused following a suboccipital exploration. Clinically, no reaction was noted and there was a favorable postoperative course, and 'autotransfusion' was evaluated as a life-saving procedure. In 1925, L.E. Davis and H. Cushing reported the first case of intraoperative autotransfusion (intraoperative blood salvage). The patient was a 42-year-old man with left occipital meningioma which, due to massive bleeding, could not be removed even by two-stage operations. In a 3rd stage operation, they could remove the tumor of 120 g totally by the aid of intraoperative replacement using 600 ml autologous blood collected with a home-made suction apparatus. No adverse side-effect was noted. This procedure was performed for 23 cases and it revealed beneficial effects except in one case. In ten of the cases, the procedure was estimated as a life-saving treatment. At the present time, the appropriate use of the patients' blood for transfusion therapy is recommended due to a shortage of donors. The use of autologous blood could play a key role, not only in saving the homologous blood supply, but also in avoiding complications encountered in conventional transfusion treatments. The methods of 'Autotransfusion' originated from operations in the neurosurgical area. On that account, it is desirable that neurosurgeons should be concerned with this subject even now.

  7. Autologous Bone Graft in Foot and Ankle Surgery.

    PubMed

    Miller, Christopher P; Chiodo, Christopher P

    2016-12-01

    Bone graft is a common adjunct procedure in orthopedic surgery used for fusions, fracture repair, and the reconstruction of skeletal defects in the foot and ankle. Autologous graft, or autograft, involves the transport of bone from a donor site to another location in the same patient. It is considered by many to be the gold standard of bone grafting, as it is provides all biologic factors required for functional graft. Further, autograft is 100% histocompatible with no risk of disease transmission.

  8. [Effectiveness of topical autologous serum treatment in neurotrophic keratopathy].

    PubMed

    Guadilla, A M; Balado, P; Baeza, A; Merino, M

    2013-08-01

    To evaluate the effectiveness of 20% autologous serum as a treatment for neurotrophic keratopathy. A longitudinal, observational and descriptive study was performed on 19 patients (22 eyes) with neurotrophic keratopathy in different stages of Mackie's classification. The following variables were evaluated on the first visit, and then 4 months later: best corrected visual acuity (BCVA), subjective patient symptomatology (faces scale), Schirmer's test without anesthesia (mm), tear film break-up time (BUT) (sg) and healing of the epithelial defect (weeks). The Wilcoxon signed-rank test was used for the statistical analysis of the data. A symptomatic improvement was observed in 100% of the cases, and a 71% improvement in best corrected visual acuity (P<.05). There was also a statistically significant improvement in the Schirmer's test and BUT (P<.05). Healing of epithelial defect occurred in 71% of the cases within 6 weeks, and in 91% of the cases within 12 weeks of treatment. The remaining 9% of the cases that did not heal had a grade 3 neurotrophic keratopathy. The use of 20% autologous topical serum represents an effective treatment for grades 1 and 2 neurotrophic keratopathy, but is an insufficient treatment for a grade 3 keratopathy. In cases where there is a significant loss of tissue, the application of a higher concentration of autologous serum, or platelet-rich derivatives, or plasma rich in growth factors, may be more effective than the application of 20% autologous serum, due to their greater effect on cell proliferation. Copyright © 2011 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  9. Autologous Rib Grafts in the Management of the Crooked Nose.

    PubMed

    Porter, Paul; Kriet, J David; Humphrey, Clinton D

    2015-06-01

    Rhinoplasty is arguably one of the most challenging procedures a facial plastic surgeon performs. Numerous techniques have been developed since the inception of rhinoplasty to aid in correction of aesthetic and functional issues. Congenital, iatrogenic, and traumatic etiologies can all lead to a crooked nose. Autologous rib or costal cartilage grafting is a powerful tool that can aid the surgeon in successful correction of the crooked nose. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  10. Antibodies: Immunoconjugates and autologous cellular therapy in acute lymphoblastic leukemia.

    PubMed

    Advani, Anjali

    2015-01-01

    Using a case study of a 57-year-old man with relapsed/refractory precursor-B (pre-B) acute lymphoblastic leukemia (ALL), this review discusses treatment with immunoconjugates and autologous therapy in acute ALL. Three therapies--blinatumomab, inotuzumab, and CAR T cells--are considered here, each with advantages in specific clinical situations. These therapies represent some of the exciting advances that have been made in the treatment of ALL over the last several years.

  11. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  12. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  13. Generative surgery of cultured autologous auricular chondrocytes for nasal augmentation.

    PubMed

    Yanaga, Hiroko; Imai, Keisuke; Yanaga, Katsu

    2009-11-01

    Conventional treatment for nasal augmentation utilizes autologous grafts, allografts, or synthetic implants such as silicon implants. Silicon implants could protrude/expose or induce nasal bone resorption. Autologous grafts are usually associated with donor site morbidity and the volume of harvested tissue is limited. We had developed a new method for nasal augmentation using cultured autologous chondrocytes (CAC). The current report presents the results of a study using that method with a larger number of patients and an improved graft technique for the nasal tip. Approximately 1 cm2 of cartilage was harvested from the auricular concha and treated with collagenase, and then chondrocytes were obtained. In our multilayer culture system the chondrocytes formed immature cartilaginous tissues with a gelatinous chondroid matrix. They were injection-grafted into the subcutaneous pocket of the nasal dorsum. The chondrocytes with a gelatinous chondroid matrix change from a soft gel to hard neocartilage tissue within 2 to 3 weeks and then stabilize. The authors have used this procedure over a 6-year period on 75 cases: 58 secondary augmentation rhinoplasties following silicon implantation and 17 primary augmentation cases. The results have been satisfactory and long-lasting. Grafting of CAC is an optional method for nasal augmentation and could be used for a wide range of facial augmentation cases.

  14. AUTOLOGOUS IMMUNE COMPLEX NEPHRITIS INDUCED WITH RENAL TUBULAR ANTIGEN

    PubMed Central

    Glassock, Richard J.; Edgington, Thomas S.; Watson, J. Ian; Dixon, Frank J.

    1968-01-01

    The pathogenetic mechanism involved in a form of experimental allergic glomerulonephritis induced by immunization of rats with renal tubular antigen has been investigated. A single immunization with less than a milligram of a crude renal tubular preparation, probably containing less than 25 µg of the specific nephritogenic antigen, is effective in the induction of this form of chronic membranous glomerulonephritis. In the nephritic kidney autologous nephritogenic tubular antigen is found in the glomerular deposits along with γ-globulin and complement. When large amounts of antigen are injected during induction of the disease the exogenous immunizing antigen can also be detected in the glomerular deposits. It appears that this disease results from the formation of circulating antibodies capable of reacting with autologous renal tubular antigen(s) and the deposition of these antibodies and antigen(s) plus complement apparently as immune complexes in the glomeruli. This pathogenetic system has been termed an autologous immune complex disease and the resultant glomerulonephritis has been similarly designated. PMID:4169966

  15. Outpatient Autologous Stem Cell Transplantation for Patients With Myeloma.

    PubMed

    Paul, Thomas M; Liu, Stephen V; Chong, Elise A; Luger, Selina M; Porter, David L; Schuster, Stephen J; Tsai, Donald E; Nasta, Sunita D; Loren, Alison; Frey, Noelle; Perl, Alexander; Cohen, Adam D; Weiss, Brendan M; Stadtmauer, Edward A; Vogl, Dan T

    2015-09-01

    High-dose melphalan with autologous stem cell support improves survival for patients with myeloma. For selected patients, we have been using a protocol of short hospitalization, discharging patients to home with careful outpatient monitoring in the office, which we hypothesized would reduce complications and utilization of inpatient beds. We reviewed 301 initial autologous transplants for myeloma, categorized as brief stay (≤ 4 days, 82 patients) or prolonged stay (≥ 5 days, 219 patients). Selection for a brief stay was determined by clinical characteristics, availability of caregivers at home, distance from our medical center, and patient preference. Within the brief stay population, 67% required readmission before day + 100, but this group still had fewer cumulative hospital days (9 vs. 18, P < .0001). There were fewer documented infections among brief stay patients (22% vs. 46% P < .001) and fewer admissions to intensive care units (0% vs. 5.9%, P = .02). The groups had similar rates of bleeding (1.2% vs. 1.4% P = 1.0) and thrombosis (3.7% vs. 4.6% P = 1.0). No patients in the brief stay group died within 100 days, compared with mortality of 1.8% (P = .6) in the prolonged stay group. Carefully selected patients receiving an autologous stem cell transplant for treatment of myeloma can be managed with a brief initial hospitalization and outpatient follow-up, with low morbidity and mortality. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Clinical Allogeneic and Autologous Islet Cell Transplantation: Update

    PubMed Central

    2011-01-01

    Islet cell transplantation is categorized as a β-cell replacement therapy for diabetic patients who lack the ability to secrete insulin. Allogeneic islet cell transplantation is for the treatment of type 1 diabetes, and autologous islet cell transplantation is for the prevention of surgical diabetes after a total pancreatectomy. The issues of allogeneic islet cell transplantation include poor efficacy of islet isolation, the need for multiple donor pancreata, difficulty maintaining insulin independence and undesirable side effects of immunosuppressive drugs. Those issues have been solved step by step and allogeneic islet cell transplantation is almost ready to be the standard therapy. The donor shortage will be the next issue and marginal and/or living donor islet cell transplantation might alleviate the issue. Xeno-islet cell transplantation, β-cell regeneration from human stem cells and gene induction of the naïve pancreas represent the next generation of β-cell replacement therapy. Autologous islet cell transplantation after total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain is the standard therapy, even though only limited centers are able to perform this treatment. Remote center autologous islet cell transplantation is an attractive option for hospitals performing total pancreatectomies without the proper islet isolation facilities. PMID:21785738

  17. Alignment of the Fibrin Network Within an Autologous Plasma Clot.

    PubMed

    Gessmann, Jan; Seybold, Dominik; Peter, Elvira; Schildhauer, Thomas Armin; Köller, Manfred

    2016-01-01

    Autologous plasma clots with longitudinally aligned fibrin fibers could serve as a scaffold for longitudinal axonal regrowth in cases of traumatic peripheral nerve injuries. Three different techniques for assembling longitudinally oriented fibrin fibers during the fibrin polymerization process were investigated as follows: fiber alignment was induced by the application of either a magnetic field or-as a novel approach-electric field or by the induction of orientated flow. Fiber alignment was characterized by scanning electron microscopy analysis followed by image processing using fast Fourier transformation (FFT). Besides FFT output images, area xmin to xmax, as well as full width at half maximum (FWHM) of the FFT graph plot peaks, was calculated to determine the relative degree of fiber alignment. In addition, fluorescently labeled human fibrinogen and mesenchymal stem cells (MSCs) were used to visualize fibrin and cell orientation in aligned and nonaligned plasma clots. Varying degrees of fiber alignment were achieved by the three different methods, with the electric field application producing the highest degree of fiber alignment. The embedded MSCs showed a longitudinal orientation in the electric field-aligned plasma clots. The key feature of this study is the ability to produce autologous plasma clots with aligned fibrin fibers using physical techniques. This orientated internal structure of an autologous biomaterial is promising for distinct therapeutic applications, such as a guiding structure for cell migration and growth dynamics.

  18. Autologous Matrix-Induced Chondrogenesis in the Knee: A Review.

    PubMed

    Lee, Yee Han Dave; Suzer, Ferzan; Thermann, Hajo

    2014-07-01

    Autologous matrix-induced chondrogenesis (AMIC) is a 1-step cartilage restoration technique that combines microfracture with the use of an exogenous scaffold. This matrix covers and mechanically stabilizes the clot. There have been an increasing number of studies performed related to the AMIC technique and an update of its use and results is warranted. Using the PubMed database, a literature search was performed using the terms "AMIC" or "Autologous Matrix Induced Chondrogenesis." A total of 19 basic science and clinical articles were identified. Ten studies that were published on the use of AMIC for knee chondral defects were identified and the results of 219 patients were analyzed. The improvements in Knee Injury and Osteoarthritis Outcome Score, International Knee Documentation Committee Subjective, Lysholm and Tegner scores at 2 years were comparable to the published results from autologous chondrocyte implantation (ACI) and matrix ACI techniques for cartilage repair. Our systematic review of the current state of the AMIC technique suggests that it is a promising 1-stage cartilage repair technique. The short-term clinical outcomes and magnetic resonance imaging results are comparable to other cell-based methods. Further studies with AMIC in randomized studies versus other repair techniques such as ACI are needed in the future.

  19. Procedure, applications, and outcomes of autologous fat grafting.

    PubMed

    Simonacci, Francesco; Bertozzi, Nicolò; Grieco, Michele Pio; Grignaffini, Eugenio; Raposio, Edoardo

    2017-08-01

    To systematically review the procedure, applications, and outcomes of autologous fat grafting, a promising technique with various clinical applications. Literature review of publications concerning autologous fat grafting. Since its introduction, lipofilling has become increasingly popular; however, its results are variable and unpredictable. Several modifications have been made to the procedures of fat harvesting, processing, and injecting. Surgical excision and low negative-pressure aspiration with large-bore cannulas minimize adipocyte damage during fat harvesting. The "wet" method of fat harvesting involves fluid injection at the donor site and facilitates lipoaspiration while minimizing pain and ecchymosis. For fat processing, centrifugation at a low speed is preferable to high-speed centrifugation, gravity separation or filtration. Fat injection at the recipient site should be performed using small-gauge cannulas in a fanning out pattern over multiple sessions, rather than a single session. Fat grafts exhibit not only dermal filler properties but also regenerative potential owing to the presence of stem cells in fat tissue. Thus, the clinical applications of autologous fat grafting include correction of secondary contour defects after breast reconstruction, release of painful scar contractures, and treatment of burn scars and radiodermatitis. Lipofilling is also used in aesthetic surgery, such as facial and hand rejuvenation, augmentation rhinoplasty, and breast and gluteal augmentation. The complications of lipofilling are minimal and include bruising, swelling, pain, infection, necrosis, and calcification. Lipofilling is a low-risk procedure that can be used to correct soft-tissue defects in the face, trunk, and extremities, with minimal discomfort for patients.

  20. [Therapeutic intensification and autologous stem cell transplantation in autoimmune diseases].

    PubMed

    Marjanovic, Z; Gerber, I; Toledano, C; Hen-Solal, J; Damade, R; de Saint-Cyr, I; Sarrot-Reynauld, F; Ilié, D; Daneshpouy, M; Mounier, N; Ruivard, M; Tyndall, C; Vidal, E; Quere, I; Durand, J-M; Constans, J; Farge, D

    2005-02-26

    THE PATHOPHYSIOLOGY of most autoimmune diseases is often poorly understood. EXPERIMENTAL CONSIDERATIONS and clinical experience suggest that high doses immunoablation followed by stem cell transplantation is a therapeutic option to consider for certain severe autoimmune disorders. THE CONCEPT OF RESTORING NORMAL IMMUNE REACTIVITY must in part br true since current results of 466 transplants (445 autologous, 21 allogeneic) patients suffering from various autoimmune diseases show a beneficial outcome in approximately 2/3 of the patients. TO IMPROVE THE EFFICACY AND SAFETY OF SUCH AN AGGRESSIVE PROCEDURE in patients with potentially affected vital organs by the underlying autoimmune disease, it is especially important to follow international consensus guidelines and to centrally collect clinical data for in depth analysis in the EBMT International Stem Cell Project for Autoimmune Disease in Basel, Switzerland. PHASE III STUDIES ARE RUNNING FOR SYSTEMIC SCLEROSIS (Astis, Autologous Stem cell Transplantation International Rheumatoid Arthritis Trial) started in 2003. A STUDY PROJECT IS PLANNED FOR MULTIPLE SCLEROSIS (Astims, Autologous Stem cell Transplantation International Multiple Sclerosis).

  1. Osteogenicity of autologous bone transplants in the goat.

    PubMed

    Kruyt, Moyo C; Dhert, Wouter J A; Oner, Cumhur; van Blitterswijk, Clemens A; Verbout, Abraham J; de Bruijn, Joost D

    2004-02-27

    Little is known about the specific mechanisms that make autologous graft bone (AG) superior to the current alternatives. A potential mechanism is the active bone formation by the osteoprogenitor cells within the AG. However, whether these cells survive the transplantation is questionable, especially in nonvascularized, clinically sized grafts. In the present study, we investigated the role of viability in AG implanted ectopically and orthotopically in the goat. Eight goats were operated on twice. At the first operation, pieces of vital or devitalized autologous cortical bone were implanted in the paraspinal muscles. Eight weeks later, corticocancellous plugs were taken from the femoral condyles, morselized, and reimplanted as either vital or devitalized orthotopic grafts. The goats received fluorochrome labels at 5, 7, and 9 weeks after the first operation. At 12 weeks, the goats were killed, and the samples were examined histologically. Ectopically, new bone had formed in both the vital and devitalized grafts. In the vital grafts, all three fluorochrome labels were present, indicating an early osteogenic mechanism. Within the devitalized grafts, only the 9-week label was observed. Histomorphometry indicated significantly more new bone in the vital grafts (10.3% vs. 1.7% in the devitalized grafts, P <0.01). Orthotopically, both vital and devitalized grafts showed new bone. Again, graft viability was advantageous in terms of new bone formation (14.5% vs. 9.3%, P <0.02). The cells inside the autologous bone transplants most likely survived transplantation and were capable of initiating and sustaining new bone formation.

  2. Tissue-Engineered Autologous Grafts for Facial Bone Reconstruction

    PubMed Central

    Bhumiratana, Sarindr; Bernhard, Jonathan C.; Alfi, David M.; Yeager, Keith; Eton, Ryan E.; Bova, Jonathan; Shah, Forum; Gimble, Jeffrey M.; Lopez, Mandi J.; Eisig, Sidney B.; Vunjak-Novakovic, Gordana

    2016-01-01

    Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care—the use of bone harvested from another region in the body—has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, without bone morphogenic proteins, using native bovine bone matrix and a perfusion bioreactor for the growth and transport of living grafts. The ramus-condyle unit (RCU), the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatan minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material, and crafted it into an anatomically correct shape using image-guided micromilling, to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either non-seeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering. PMID:27306665