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Sample records for rna transfected autologous

  1. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors

    PubMed Central

    Heiser, Axel; Coleman, Doris; Dannull, Jens; Yancey, Donna; Maurice, Margaret A.; Lallas, Costas D.; Dahm, Philipp; Niedzwiecki, Donna; Gilboa, Eli; Vieweg, Johannes

    2002-01-01

    Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell–mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA–transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer. PMID:11828001

  2. Immunization of HIV-1-Infected Persons With Autologous Dendritic Cells Transfected With mRNA Encoding HIV-1 Gag and Nef: Results of a Randomized, Placebo-Controlled Clinical Trial

    PubMed Central

    Kwon, Douglas S.; Macklin, Eric A.; Shopis, Janet R.; McLean, Anna P.; McBrine, Nicole; Flynn, Theresa; Peter, Lauren; Sbrolla, Amy; Kaufmann, Daniel E.; Porichis, Filippos; Walker, Bruce D.; Bhardwaj, Nina; Barouch, Dan H.; Kavanagh, Daniel G.

    2016-01-01

    Background: HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy. Methods: Fifteen participants were randomized 2:1 to receive intradermal immunization with HIV-1 Gag- and Nef-transfected DCs (vaccine) or mock-transfected DCs (placebo) at weeks 0, 2, 6, and 10. All participants also received DCs pulsed with keyhole limpet hemocyanin (KLH) to assess whether responses to a neo-antigen could be induced. Results: After immunization, there were no differences in interferon-gamma enzyme-linked immunospot responses to HIV-1 Gag or Nef in the vaccine or placebo group. CD4 proliferative responses to KLH increased 2.4-fold (P = 0.026) and CD8 proliferative responses to KLH increased 2.5-fold (P = 0.053) after vaccination. There were increases in CD4 proliferative responses to HIV-1 Gag (2.5-fold vs. baseline, 3.4-fold vs. placebo, P = 0.054) and HIV-1 Nef (2.3-fold vs. baseline, 6.3-fold vs. placebo, P = 0.009) among vaccine recipients, but these responses were short-lived. Conclusion: Immunization with DCs transfected with mRNA encoding HIV-1 Gag and Nef did not induce significant interferon-gamma enzyme-linked immunospot responses. There were increases in proliferative responses to HIV-1 antigens and to a neo-antigen, KLH, but the effects were transient. Dendritic cell vaccination should be optimized to elicit stronger and long-lasting immune responses for this strategy to be effective as an HIV-1 therapeutic vaccine. PMID:26379068

  3. Immune Monitoring Using mRNA-Transfected Dendritic Cells.

    PubMed

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by mRNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA. PMID:27236804

  4. Optimization of Transfection Conditions for siRNA Screening.

    PubMed

    Montoya, Justin J; Azorsa, David O

    2016-01-01

    RNAi screening of mammalian cells is often performed using siRNAs and cationic lipids as transfection reagents. Efficiency of transfection depends on growth characteristics of the cells and the cationic lipid used. With a large selection of cationic lipids available, it can often be difficult to select the optimal lipid and lipid:siRNA (vol:wt) ratio. Here, we describe the process of optimizing siRNA transfection conditions for efficient reverse transfection of mammalian cells using specific positive and negative siRNA controls. PMID:27581281

  5. Transfecting RNA quadruplexes results in few transcriptome perturbations

    PubMed Central

    Sanders, Phil G.T.; Cotterell, James; Sharpe, James; Isalan, Mark

    2013-01-01

    Guanine-rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. Previous studies showed that transfecting G-quadruplex DNA oligonucleotides inhibits proliferation in many cancer cell lines and can induce apoptosis. However, little is known about the effects of transfecting RNA quadruplexes. In this study, we transfected a G-quadruplex RNA oligonucleotide (GqRNA) into HEK293T cells and observed that it did not alter cell viability. Subsequent transcriptome expression profiling revealed that only two genes, EGR1 and FOS, were significantly altered in the presence of GqRNA (upregulated 2- to 4-fold). Sequence analysis showed that both genes contained putative quadruplex sequences (PQS) in their 3′-UTRs, immediately adjacent to the stop codons. Transfection of the EGR1 PQS as an RNA oligonucleotide also caused an increase in EGR1 expression. Similar motifs are found in a variety of genomes, but are relatively rare and have been missed by previous annotations. A bioinformatic analysis revealed stop codon-proximal enrichment of such motifs compared with the rest of the 3′-UTR, although these genes were not affected by RNA quadruplex transfection, and their function remains unknown. Overall, transfecting RNA quadruplexes results in relatively few alterations in gene expression. PMID:23235467

  6. RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

    PubMed Central

    Liao, C L; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur

  7. Acoustic Liquid Handling for Rapid siRNA Transfection Optimization.

    PubMed

    Xiao, Andrew S; Lightcap, Eric S; Bouck, David C

    2015-09-01

    Gene knockdown by small interfering RNA (siRNA) has been used extensively to investigate the function of genes in targeted and genome-wide studies. One of the primary challenges of siRNA studies of any scale is to achieve sufficient gene knockdown to produce the biological changes that lead to measurable phenotypes. Reverse, lipid-based transfection efficiency minimally requires the optimization of the following parameters: cell number, knockdown duration, siRNA oligonucleotide concentration, type/brand of transfection lipid, and transfection lipid concentration. In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues. PMID:25924619

  8. 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

  9. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

    RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis. PMID:27581282

  10. Allogeneic mRNA-based electrotransfection of autologous dendritic cells and specific antitumor effects against osteosarcoma in rats.

    PubMed

    Yu, Zhe; Qian, Jixian; Wu, Jiachang; Gao, Jie; Zhang, Minghua

    2012-12-01

    Vaccination with dendritic cells (DCs) transfected with tumor-derived mRNA antigen has emerged as a promising strategy for generating protective immunity in mammals. However, the integration of allogeneic osteosarcoma mRNA and autologous DCs has not been fully examined. This study was designed to investigate the antitumor effects of tumor vaccine produced by autologous DCs transfected of allogeneic osteosarcoma mRNA through electroporation in tumor-bearing rats model. In the present study, extraction of Wistar rat tumor mRNA was performed as a two-step procedure. First, total RNA was extracted by use of Trizol; then, mRNA purification was performed by use of polyT-coated magnetic beads. Then, we transfected the allogeneic-derived tumor mRNA to Sprague-Dawley (SD) rat bone marrow-derived DCs through electroporation. The tumor vaccine was applied to tumor-bearing rats model, and the specific antitumor effects of the tumor vaccine were observed. The immunization using autologous DCs electrotransfected with allogeneic osteosarcoma total RNA induced specific CTL responses, which were statistically significant (P < 0.05), and the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking MHC class I molecules. In in vivo experiments, 70 % of the rats immunized with allogeneic osteosarcoma RNA transfected to DCs were typically able to reject tumor challenge and remained tumor-free. Vaccinated survivors developed long immunological memory and were able to reject a subsequent rechallenge with the same tumor cells but not a syngeneic unrelated tumor line. In the present study, we demonstrated that allogeneic tumor mRNA isolated from rat osteosarcoma cell line could be applied to produce tumor vaccine inducing specific antitumor effects, especially in DC-based immunotherapy strategy. This study also provides the foundations for an effective and broadly applicable treatment to a wide range of cancer indications for which tumor-associated antigens

  11. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

    PubMed Central

    Jensen, Kirsty; Anderson, Jennifer A.; Glass, Elizabeth J.

    2014-01-01

    The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA. PMID:24598124

  12. Transfection of microRNA Mimics Should Be Used with Caution.

    PubMed

    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

  13. 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

  14. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite.

    PubMed

    Zhang, Gen; He, Li-Sheng; Wong, Yue Him; Yu, Li; Qian, Pei-Yuan

    2015-08-01

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in 'double transfections', in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments. PMID:26113139

  15. Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems.

    PubMed

    Selmeczi, David; Hansen, Thomas Steen; Met, Özcan; Svane, Inge Marie; Larsen, Niels B

    2016-01-01

    Electroporation is well established for transient mRNA transfection of many mammalian cells, including immune cells such as dendritic cells used in cancer immunotherapy. Therapeutic application requires methods to efficiently electroporate and transfect millions of immune cells in a fast process with high cell survival. Continuous flow of suspended dendritic cells through a channel incorporating spatially separated microporous meshes with a synchronized electrical pulsing sequence can yield dendritic cell transfection rates of >75 % with survival rates of >90 %. This chapter describes the instrumentation and methods needed for the efficient transfection by electroporation of millions of dendritic cells in one continuous flow process. PMID:27236798

  16. A peptidomimetic siRNA transfection reagent forhighly effectivegene silencing

    SciTech Connect

    Utku, Yeliz; Dehan, Elinor; Ouerfelli, Ouathek; Piano, Fabio; Zuckermann, Ronald N.; Pagano, Michele; Kirshenbaum, Kent

    2006-05-17

    RNA interference (RNAi) techniques hold forth great promisefor therapeutic silencing of deleterious genes. However, clinicalapplications of RNAi require the development of safe and efficientmethods for intracellular delivery of small interfering RNA (siRNA)oligonucleotides specific to targeted genes. We describe the use of alipitoid, a cationic oligopeptoid phospholipid conjugate, for non-viraltransfection of synthetic siRNA oligos in cell culture. Thispeptidomimetic delivery vehicle allows for efficient siRNA transfectionin a variety of human cell lines with negligible toxicity and promotesextensive downregulation of the targeted genes at both the protein andthe mRNA level. We compare the lipitoid reagent to a standard commercialtransfection reagent. The lipitoid is highly efficient even in primaryIMR-90 human lung fibroblasts in which other commercial reagents aretypically ineffective.

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

    PubMed Central

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

    2014-01-01

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

  18. 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

  19. 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. PMID:26974323

  20. 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

  1. 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

  2. Preconditioning Vaccine Sites for mRNA-Transfected Dendritic Cell Therapy and Antitumor Efficacy.

    PubMed

    Batich, Kristen A; Swartz, Adam M; Sampson, John H

    2016-01-01

    Messenger RNA (mRNA)-transfected dendritic cell (DC) vaccines have been shown to be a powerful modality for eliciting antitumor immune responses in mice and humans; however, their application has not been fully optimized since many of the factors that contribute to their efficacy remain poorly understood. Work stemming from our laboratory has recently demonstrated that preconditioning the vaccine site with a recall antigen prior to the administration of a dendritic cell vaccine creates systemic recall responses and resultantly enhances dendritic cell migration to the lymph nodes with improved antitumor efficacy. This chapter describes the generation of murine mRNA-transfected DC vaccines, as well as a method for vaccine site preconditioning with protein antigen formulations that create potent recall responses. PMID:27076169

  3. 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

  4. Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

    PubMed

    Sohn, Dae-Hee; Sohn, Hyun-Jung; Lee, Hyun-Joo; Lee, Seon-Duk; Kim, Sueon; Hyun, Seung-Joo; Cho, Hyun-Il; Cho, Seok-Goo; Lee, Suk-Kyeong; Kim, Tai-Gyu

    2015-01-01

    An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8(+) and CD4(+) T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8(+) and CD4(+) T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4(+) T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+) T cells was significantly correlated with that of CD8(+) T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+) T cell responses showed more significant changes than CD8(+) T cell responses. CD8(+) and CD4(+) T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4(+) T cells secreted significantly higher cytokine levels than did CD8(+) T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases. PMID:26023769

  5. Oxygen-glucose deprivation of neurons transfected with toll-like receptor 3-siRNA: Determination of an optimal transfection sequence

    PubMed Central

    Cui, Guiyun; Wang, Xiaopeng; Ye, Xinchun; Zu, Jie; Zan, Kun; Hua, Fang

    2013-01-01

    Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ischemia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxygen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfering RNA (siRNA)-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like receptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation. PMID:25206644

  6. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors.

    PubMed

    Wang, Jie; Lu, Ze; Yeung, Bertrand Z; Wientjes, M Guillaume; Cole, David J; Au, Jessie L-S

    2014-03-28

    Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer. PMID:24462901

  7. Paclitaxel tumor priming promotes delivery and transfection of intravenous lipid-siRNA in pancreatic tumors.

    PubMed

    Wang, Jie; Lu, Ze; Wang, Junfeng; Cui, Minjian; Yeung, Bertrand Z; Cole, David J; Wientjes, M Guillaume; Au, Jessie L-S

    2015-10-28

    The major barrier for using small interfering RNA (siRNA) as cancer therapeutics is the inadequate delivery and transfection in solid tumors. We have previously shown that paclitaxel tumor priming, by inducing apoptosis, expands the tumor interstitial space, improves the penetration and dispersion of nanoparticles and siRNA-lipoplexes in 3-dimensional tumor histocultures, and promotes the delivery and transfection efficiency of siRNA-lipoplexes under the locoregional setting in vivo (i.e., intraperitoneal treatment of intraperitoneal tumors). The current study evaluated whether tumor priming is functional for systemically delivered siRNA via intravenous injection, which would subject siRNA to several additional delivery barriers and elimination processes. We used the same pegylated cationic (PCat)-siRNA lipoplexes as in the intraperitoneal study to treat mice bearing subcutaneous human pancreatic Hs766T xenograft tumors. The target gene was survivin, an inducible chemoresistance gene. The results show single agent paclitaxel delayed tumor growth but also significantly induced the survivin protein level in residual tumors, whereas addition of PCat-siSurvivin completely reversed the paclitaxel-induced survivin and enhanced the paclitaxel activity (p<0.05). In comparison, PCat-siSurvivin alone did not yield survivin knockdown or antitumor activity, indicating the in vivo effectiveness of intravenous siRNA-mediated gene silencing requires paclitaxel cotreatment. Additional in vitro studies showed that paclitaxel promoted the cytoplasmic release of siGLO, a 22 nucleotide double-stranded RNA that has no mRNA targets, from its PCat lipoplex and/or endosomes/lysosomes. Taken together, our earlier and current data show paclitaxel tumor priming, by promoting the interstitial transport and cytoplasmic release, is critical to promote the delivery and transfection of siRNA in vivo. In addition, because paclitaxel has broad spectrum activity and is used to treat multiple types

  8. Reduction of EGF receptor levels in human tumor cells transfected with an antisense RNA expression vector

    SciTech Connect

    Yamada, Hirotomo; Koizumi, Shinji; Kimura, Masami ); Shimizu, Nobuyoshi )

    1989-09-01

    An expression vector was constructed from part of pSV2neo with the 3{prime}-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction cell-surface EGF receptor in response to ZnSO{sub 4}. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

  9. Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

    PubMed Central

    Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W.

    2015-01-01

    Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases. PMID:25923429

  10. Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity.

    PubMed

    Fujii, Shin-ichiro; Goto, Akira; Shimizu, Kanako

    2009-04-30

    The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of alpha-galactosylceramide (alpha-GalCer)-loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with alpha-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)-mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with alpha-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases. PMID:19164596

  11. Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells.

    PubMed

    Bettinger, T; Carlisle, R C; Read, M L; Ogris, M; Seymour, L W

    2001-09-15

    Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells. PMID:11557821

  12. 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. PMID:24002693

  13. 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

  14. Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage.

    PubMed

    Boros, Gábor; Karikó, Katalin; Muramatsu, Hiromi; Miko, Edit; Emri, Eszter; Hegedűs, Csaba; Emri, Gabriella; Remenyik, Éva

    2016-01-01

    In vitro-synthesized mRNA containing nucleoside modifications has great therapeutical potential to transiently express proteins with physiological importance. One such protein is photolyase which rapidly removes UV-induced DNA damages, but this enzyme is absent in humans. Here, we apply a novel mRNA-based platform to achieve functional nonhuman photolyase production in cultured human keratinocytes. Transfection of nucleoside-modified mRNA encoding photolyase leads to accelerated repair of DNA photolesions in human keratinocytes. PMID:27236802

  15. Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2.

    PubMed

    Idorn, Manja; Thor Straten, Per; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Adoptive T-cell therapy based on the infusion of patient's own immune cells after ex vivo culturing is among the most potent forms of personalized treatment among recent clinical developments for the treatment of cancer. However, despite high rates of successful initial clinical responses, only about 20 % of patients with metastatic melanoma treated with tumor-infiltrating lymphocytes (TILs) enter complete and long-term regression, with the majority either relapsing after initial partial regression or not benefiting at all. Previous studies have shown a positive correlation between the number infused T cells migrating to the tumor and the clinical response, but also that only a small fraction of adoptively transferred T cells reach the tumor site. In this chapter, we describe a protocol for transfection of TILs with mRNA encoding the chemokine receptor CXCR2 transiently redirecting and improving TILs migration toward tumor-secreted chemokines in vitro. PMID:27236805

  16. 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

  17. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    PubMed

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis. PMID:21861063

  18. Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

    PubMed

    Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

    2013-01-01

    Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy. PMID:23296935

  19. MicroRNA-338 and microRNA-21 co-transfection for the treatment of rat sciatic nerve injury.

    PubMed

    Wang, Jianyong; Muheremu, Aikeremujiang; Zhang, Ming; Gong, Kai; Huang, Chuyi; Ji, Yuchen; Wei, Yujun; Ao, Qiang

    2016-06-01

    The objective of this study is to find if co-transfecting microRNA-338 and microRNA-21 into the neurons in the spinal cord can promote functional recovery after peripheral nerve injury in rats. Animals were divided into three groups: 20 animals in the GFP control vector group (group A), 20 animals in the GFP experimental vector group (group B) and ten animals in the normal control group. Right sciatic nerves of animals in groups A and B were transected and were bridged with collagen nerve conduits with 10 mm distance between the stumps. 3 µl GFP control vector or 3 µl lentiviral vectors encoding the sequence of microRNA-338 and microRNA-21 were injected in the conduit. 8 weeks after the surgery, the treatment effect was evaluated by functional analysis, electrophysiological analysis, immunohistochemical analysis as well as transmitting electronic microscope observations in all the rats. Animals treated with microRNA-338 and microRNA-21 showed significantly better recovery than GFP control group animals by means of functional analysis (Sciatic nerve index -47.7 ± 2.5 vs -59.4 ± 3.7), electrophysiological analysis (Conduction velocity 20.5 ± 2.8 vs 10.5 ± 1.4 m/s), ratio of wet weight of the gastrocnemius muscles (0.83 ± 0.03 vs 0.55 ± 0.06), axon diameter (5.0 ± 1.8 µm vs 4.0 ± 2.2), myelin sheath thickness (1.4 ± 0.43 vs 0.80 ± 0.31 µm) and G-ratio (0.80 ± 0.06 vs 0.75 ± 0.04). Lentiviral vectors encoding microRNA 338 and 21 might be explored in the future as potential therapeutic intervention to promote nerve regeneration. PMID:26909749

  20. 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.

  1. 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

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

    PubMed

    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 Ca(2+) 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 (G(oA) 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

  3. 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. PMID:26690650

  4. Transfection of shRNA-encoding Minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells

    PubMed Central

    Zhao, N; Fogg, J M; Zechiedrich, L; Zu, Y

    2011-01-01

    This work illustrates the utility of Minivector DNA, a non-viral, supercoiled gene therapy vector incorporating short hairpin RNA from an H1 promoter. Minivector DNA is superior to both plasmid DNA and small interfering RNA (siRNA) in that it has improved biostability while maintaining high cell transfection efficiency and gene silencing capacity. Minivector DNAs were stable for over 48 h in human serum, as compared with only 0.5 and 2 h for siRNA and plasmid, respectively. Although all three nucleic acids exhibited similar transfection efficiencies in easily transfected adhesion fibroblasts cells, only Minivector DNAs and siRNA were capable of transfecting difficult-to-transfect suspension lymphoma cells. Minivector DNA and siRNA were capable of silencing the gene encoding anaplastic lymphoma kinase, a key pathogenic factor of human anaplastic large cell lymphoma, and this silencing caused inhibition of the lymphoma cells. Based on these results, Minivector DNAs are a promising new gene therapy tool. PMID:20962872

  5. 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. PMID:27321701

  6. Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells.

    PubMed

    Hansson, Magnus L; Albert, Silvia; González Somermeyer, Louisa; Peco, Rubén; Mejía-Ramírez, Eva; Montserrat, Núria; Izpisua Belmonte, Juan Carlos

    2015-02-27

    Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional. PMID:25555917

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

    PubMed

    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 (51)Cr-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 (51)Cr 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

  8. 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

  9. 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

  10. Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: stability and interactions with the core RNAi machinery.

    PubMed

    Swevers, Luc; Kolliopoulou, Anna; Li, Zheng; Daskalaki, Maria; Verret, Frederic; Kalantidis, Kriton; Smagghe, Guy; Sun, Jingchen

    2014-05-01

    While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of an in vitro culture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for an in vitro culture system for BmCPV. PMID:24636911

  11. 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

    2015-03-01

    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. PMID:25603322

  12. mRNA Transfection to Improve NK Cell Homing to Tumors.

    PubMed

    Levy, Emily R; Carlsten, Mattias; Childs, Richard W

    2016-01-01

    The ability of natural killer (NK) cells to mediate antitumor effects following adoptive transfer is dependent on their capacity to traffic to the microenvironment where tumors reside. Recent studies have shown that cytokine-activated and ex vivo-expanded NK cells lack or express at low levels homing receptors required to achieve tissue-specific tumor targeting by cells administered intravenously. In this chapter, we describe a method to enhance NK cell homing toward specific chemoattractants expressed in secondary lymphoid tissues through genetic modification of NK cells using mRNA electroporation. The method described here is scalable, cGMP-compliant, and offers a strategy to bolster the efficacy of adoptive NK cell immunotherapy for the treatment of hematological malignancies in the clinic. PMID:27177670

  13. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  14. Genome-Wide siRNA Screening Using Forward Transfection: Identification of Modulators of Membrane Trafficking in Mammalian Cells.

    PubMed

    Bexiga, Mariana G; Simpson, Jeremy C

    2016-01-01

    RNA interference (RNAi) has become an essential tool for molecular and cellular biologists to dissect cell function. In recent years its application has been extended to genome-wide studies, enabling the systematic identification of new cell regulation mechanisms and drug targets. In this chapter, a protocol for a genome-wide RNAi screen coupled to high-content microscopy is presented. Specifically we describe key features of assay design, plate layout, and a protocol for forward transfection of small interfering RNAs (siRNAs) in a 384-well plate format. As an example of its application in identifying modulators of membrane trafficking, we also provide a protocol to measure the efficacy of intracellular delivery of the B subunit of Shiga-like toxin to the Golgi complex. Finally we show an automated image analysis routine that can be used to extract single cell data from the screen, thereby providing a quantitative ranking of how a large panel of siRNAs affects this biological process. PMID:27581283

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

    PubMed

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

    2010-12-15

    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 IFNβ 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

  16. 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. PMID:27047347

  17. 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

  18. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential

    PubMed Central

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

    2013-01-01

    UVB irradiation induces harmful photochemical reactions, including formation of cyclobutane pyrimidine dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2 days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  19. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.

    PubMed

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

    2013-12-01

    UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  20. Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer

    PubMed Central

    2014-01-01

    Background We previously reported the clinical efficacy of adoptive immunotherapy (AIT) with dendritic cells (DCs) pulsed with mucin 1 (MUC1) peptide and cytotoxic T lymphocytes (CTLs). We also reported that gemcitabine (GEM) enhances anti-tumor immunity by suppressing regulatory T cells. Therefore, in the present study, we performed combination therapy with AIT and GEM for patients with unresectable or recurrent pancreatic cancer. Patients and methods Forty-two patients with unresectable or recurrent pancreatic cancer were treated. DCs were generated by culture with granulocyte macrophage colony-stimulating factor and interleukin-4 and then exposed to tumor necrosis factor-α. Mature DCs were transfected with MUC1-mRNA by electroporation (MUC1-DCs). MUC1-CTLs were induced by co-culture with YPK-1, a human pancreatic cancer cell line, and then with interleukin-2. Patients were treated with GEM, while MUC1-DCs were intradermally injected, and MUC1-CTLs were intravenously administered. Results Median survival time (MST) was 13.9 months, and the 1-year survival rate was 51.1%. Of 42 patients, one patient had complete response (2.4%), three patients had partial response (7.1%) and 22 patients had stable disease (52.4%). The disease control ratio was 61.9%. The MST and 1-year survival rate of 35 patients who received more than 1 × 107 MUC1-DCs per injection was 16.1 months and 60.3%, respectively. Liver metastasis occurred in only 5 patients among 35 patients without liver metastasis before treatment. There were no severe toxicities associated with AIT. Conclusion AIT with MUC1-DCs and MUC1-CTLs plus GEM may be a feasible and effective treatment for pancreatic cancer. PMID:24947606

  1. AB115. Effects of target gene expression on ex vivo differentiated mesenchymal stromal cells transfected by lentiviral vector with PDE5 of short hairpin RNA

    PubMed Central

    Xiao, Heng-Jun; Yan, Weixin; Chen, Jun; Gao, Xin; Zhuan, Li; Wang, Tao; Yang, Jun; Liu, Ji-Hong

    2016-01-01

    Objective To investigate the expression of PDE5 in ex vivo differentiation of gene-modified BM-MSCs using the lentiviral vector containing silencing target gene PDE5 of shRNA. Methods SD rat bone marrow-derived MSCs were separated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent culture. The third passage rat BM-MSCs were obtained, and were identified by cell surface markers with flow cytometry (FCM). The lentiviral vector carrying silencing target gene PDE5 of shRNA was constructed and then transfected into the third passage rat BM-MSCs. The gene-modified rat BM-MSCs were induced to differentiate into smooth muscle-like cells exposed to VEGF and b-FGF media in vitro. The proliferative ability of these cells was tested by cell counting kit 8 (CCK-8). Smooth muscle-like cells differentiation was assessed by immunofluorescence and then subjected to immunocytochemistry for specific markers of α-smooth muscle actin (α-SMA). Real-time quantitative PCR, immunohistochemical staining and Western blot analysis for PDE5 gene expression in differentiated smooth muscle-like cells were carried out. Results After the conducted lentiviral vector containing PDE5-shRNA was transfected into rat BM-MSCs, the expression of EGFP was detected at 24 h and it became the strongest at 72 h, which FCM showed the transfection efficiency was 91.3% at this time. The EGFP expression rates of rat BM-MSCs transfected with PDE5-shRNA at 3 d, 7 d, 14 d after transduction were 91.3%, 86.1%, and 82.7%, respectively. There was still visible green fluorescence at 28 d after transfection. The gene-modified rat BM-MSCs with PDE5-shRNA were successfully induced to differentiate into smooth muscle-like cells. Transduction of the lentiviral vector PDE5-shRNA into rat BM-MSCs led to down-regulation of PDE5. The expression of PDE5 was reduced 67.2% by PDE5-shRNA compared with the control lentiviral vector. The proliferative property of rat BM-MSCs did not

  2. RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

    PubMed Central

    Thakral, Deepshi; Joshi, Prashant; Durgapal, Hemlata; Panda, Subrat Kumar

    2014-01-01

    Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV. PMID:24505321

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

    PubMed Central

    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. PMID:26121660

  4. 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 × 10(11), 2 × 10(10), 2 × 10(9) and 2 × 10(8) 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 × 10(11) and 2 × 10(10) 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 × 10(10) 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. PMID:26381218

  5. [Corrigendum] Transient transfection of macrophage migration inhibitory factor small interfering RNA disrupts the biological behavior of oral squamous carcinoma cells.

    PubMed

    Zeng, Jie; Quan, Jingjing; Xia, Xuefeng

    2016-07-01

    Due to an inability to contact various of the contributors to this study at the time of submission and a desire to publish this work, the published article did not include the full complement of authors who should have been credited on the paper. All of the existing authors have agreed that the following authors, whose names were omitted, should also have been included as co-authors: Professor Jin Gao (now at the School of Dentistry and Oral Health, Griffith University, Queensland, Australia), who was involved with project design and revisions of the manuscript; Dr Shuyu Luo (now at the School and Hospital of Stomatology, Tianjin Medical University, Tianjin, China), who was involved in project development, data collection (Figs 3 and 6) and manuscript writing; and Dr Jianming Zhang (now at the Department of Stomatology, General Hospital of Tianjin Medical University, Tianjin, China), who was involved in project development, data collection and analysis (Fig. 4) The full author list for this paper is presented below, showing the corrected order of the authors as they should have appeared on the paper. We regret the omission of the three aforementioned authors on the published article. Note that Professor Jin Gao should be considered as the co-corresponding author (with Xuefeng Xia), and Jie Zeng and Shuyu Luo contributed equally to this study. [the original article was published in the Molecular Medicine Reports 13: 174‑180, 2015; DOI: 10.3892/mmr.2015.4525] Transient transfection of macrophage migration inhibitory factor small interfering RNA disrupts the biological behavior of oral squamous carcinoma cells Jie Zeng1*, Shuyu Luo2*, Jianming Zhang3, Jingjing Quan4, Xuefeng Xia1 and Jin Gao5 1The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150; 2School and Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, 3Department of Stomatology, General Hospital of Tianjin Medical University, Tianjin 300052; 4Guanghua

  6. Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.

    PubMed

    Ahlemeyer, Barbara; Vogt, Julia-Franziska; Michel, Vera; Hahn-Kohlberger, Petra; Baumgart-Vogt, Eveline

    2014-11-01

    The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(®) 2000, FuGENE 6, HiPerFect(®), INTERFERin™, RiboJuice™) as well as microporation using the Neon™ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70%), to the reduction of PEX5 mRNA (by 90 vs. 0-50%) and PEX5 protein levels (by 70 vs. 0-50%). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15%) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded

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

    PubMed

    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

  8. 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

  9. 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.

  10. 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

  11. Combination wt-p53 and MicroRNA-125b Transfection in a Genetically Engineered Lung Cancer Model Using Dual CD44/EGFR-targeting Nanoparticles.

    PubMed

    Talekar, Meghna; Trivedi, Malav; Shah, Parin; Ouyang, Qijun; Oka, Adwait; Gandham, Srujan; Amiji, Mansoor M

    2016-04-01

    Mutations in KRAS and p53 signaling pathways contribute to loss of responsiveness to current therapies and a decreased survival in lung cancer. In this study, we have investigated the delivery and transfection of wild-type (wt-) p53 and microRNA-125b (miR-125b) expressing plasmid DNA, in SK-LU-1 human lung adenocarcinoma cells as well as in Kras(G12D)/p53(fl/fl) (KP) genetically engineered mouse model of lung cancer. Systemic plasmid DNA delivery with dual CD44/EGFR-targeted hyaluronic acid (HA)-based nanoparticles (NPs) resulted in a 2- to 20-fold increase in wt-p53 and miR-125b gene expression in SK-LU-1 cells. This resulted in enhanced apoptotic activity as seen with increased APAF-1 and caspase-3 gene expression. Similarly, in vivo evaluations in KP mouse model indicated successful CD44/EGFR-targeted delivery. Tumor growth inhibition and apoptotic induction were also observed with (wt-p53+miR125b) combination therapy in KP tumor model. Lastly, J774.A1 murine macrophages co-cultured with transfected SK-LU-1 cells showed a 14- to 35-fold increase in the iNOS-Arg-1 ratio, supportive of previous results demonstrating a role of miR-125b in macrophage repolarization. Overall, these results show tremendous promise of wt-p53 and miR-125b gene therapy using dual CD44/EGFR-targeting HA NP vector for effective treatment of lung cancer. PMID:26686386

  12. 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.

  13. Transfected Poly(I:C) Activates Different dsRNA Receptors, Leading to Apoptosis or Immunoadjuvant Response in Androgen-independent Prostate Cancer Cells*

    PubMed Central

    Palchetti, Sara; Starace, Donatella; De Cesaris, Paola; Filippini, Antonio; Ziparo, Elio; Riccioli, Anna

    2015-01-01

    Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression. PMID:25568326

  14. Large volume flow electroporation of mRNA: clinical scale process.

    PubMed

    Li, Linhong; Allen, Cornell; Shivakumar, Rama; Peshwa, Madhusudan V

    2013-01-01

    Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3-7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency: >80% lysis at effector-target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery. PMID:23296932

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. 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

  17. 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

    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

  18. Knock-down of argonaute 2 (AGO2) induces apoptosis in myeloid leukaemia cells and inhibits siRNA-mediated silencing of transfected oncogenes in HEK-293 cells.

    PubMed

    Naoghare, Pravin K; Tak, Yu Kyung; Kim, Min Jung; Han, Eunyoung; Song, Joon Myong

    2011-10-01

    Understanding the role of oncomirs allows new insights into the development of modern therapeutic approaches for the repression of multiple oncomirs in cancer cells. At present, no suitable approach is available to repress the development of multiple oncomirs in cancer cells. Herein, we report that argonaute 2 (AGO2) could be a unique molecule to regulate the development of multiple oncomirs in cancer cells. Knock-down of AGO2 by custom-made AGO2 siRNA resulted in the induction of apoptosis in myeloid leukaemia cells (HL-60). Further investigations revealed that knock-down of AGO2 by custom-made AGO2 siRNA in HEK-293 cells resulted in silencing of the expression of target genes vascular endothelial growth factor A and histone deacetylase 2, which are known to be involved in the development of myeloid leukaemia. From these results, it can be predicted that AGO2 could regulate siRNA-mediated RNAi pathways in cancer cells. Furthermore, we investigated the possible implication of AGO2 in drug-induced apoptosis. Investigations revealed that treatment with the newly synthesized drug analogue SH-03[{(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13atetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol}] could induce AGO2-mediated apoptosis in myeloid leukaemia cells via intrinsic apoptotic pathways independent of Dicer. PMID:21535412

  19. Ultrasound mediated gene transfection

    NASA Astrophysics Data System (ADS)

    Williamson, Rene G.; Apfel, Robert E.; Brandsma, Janet L.

    2002-05-01

    Gene therapy is a promising modality for the treatment of a variety of human diseases both inherited and acquired, such as cystic fibrosis and cancer. The lack of an effective, safe method for the delivery of foreign genes into the cells, a process known as transfection, limits this effort. Ultrasound mediated gene transfection is an attractive method for gene delivery since it is a noninvasive technique, does not introduce any viral particles into the host and can offer very good temporal and spatial control. Previous investigators have shown that sonication increases transfection efficiency with and without ultrasound contrast agents. The mechanism is believed to be via a cavitation process where collapsing bubble nuclei permeabilize the cell membrane leading to increased DNA transfer. The research is focused on the use of pulsed wave high frequency focused ultrasound to transfect DNA into mammalian cells in vitro and in vivo. A better understanding of the mechanism behind the transfection process is also sought. A summary of some in vitro results to date will be presented, which includes the design of a sonication chamber that allows us to model the in vivo case more accurately.

  20. 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

  1. Mammalian cell transfection: the present and the future

    PubMed Central

    Kim, Tae Kyung

    2010-01-01

    Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective. PMID:20549496

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

    PubMed Central

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

    2015-01-01

    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. PMID:25964300

  3. Highly efficient transfection of human THP-1 macrophages by nucleofection.

    PubMed

    Maeß, Marten B; Wittig, Berith; Lorkowski, Stefan

    2014-01-01

    Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings. PMID:25226503

  4. Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

    PubMed Central

    Maeß, Marten B.; Wittig, Berith; Lorkowski, Stefan

    2014-01-01

    Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings. PMID:25226503

  5. Autologous Microvascular Breast Reconstruction

    PubMed Central

    Ramakrishnan, Venkat

    2013-01-01

    Autologous microvascular breast reconstruction is widely accepted as a key component of breast cancer treatment. There are two basic donor sites; the anterior abdominal wall and the thigh/buttock region. Each of these regions provides for a number of flaps that are successfully utilised in breast reconstruction. Refinement of surgical technique and the drive towards minimising donor site morbidity whilst maximising flap vascularity in breast reconstruction has seen an evolution towards perforator based flap reconstructions, however myocutaneous flaps are still commonly practiced. We review herein the current methods of autologous microvascular breast reconstruction. PMID:23362474

  6. 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

  7. 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. PMID:26656923

  8. 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

  9. 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

  10. On Measuring miRNAs after Transient Transfection of Mimics or Antisense Inhibitors

    PubMed Central

    Thomson, Daniel W.; Bracken, Cameron P.; Szubert, Jan M.; Goodall, Gregory J.

    2013-01-01

    The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level. PMID:23358900

  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. Autologous gastrointestinal reconstruction.

    PubMed

    Bianchi, A

    1995-02-01

    The patient with short bowel syndrome is essentially unable to absorb sufficient nutrients. This is caused by either short mucosal contact time, insufficient mucosal surface area (enterocyte mass), or a combination of the two. Management consists primarily in sustaining health and growth by intravenous nutrition and in enhancing the natural intestinal adaptation response. Surgery in the form of autologous gastrointestinal reconstruction (AGIR) is designed to redistribute the patient's own residual absorptive bowel to enhance adaptation and, possibly, to increase the absorptive mucosal surface by neomucosal growth. The alternative and ultimate fallback procedure in the management of intestinal failure is bowel transplantation, with its associated serious immunosuppression-related complications. Imaginative AGIR techniques provide new hope for the future. PMID:7728509

  13. 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. PMID:24713904

  14. 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

  15. Autologous blood storage in obstetrics.

    PubMed

    Herbert, W N; Owen, H G; Collins, M L

    1988-08-01

    Autologous transfusion, storage of one's own blood for subsequent infusion if needed, is safe and effective in a variety of scheduled operative procedures. Obstetric involvement in such programs is very limited, however. Thirty pregnant women with placenta previa or other potential complications underwent 55 phlebotomies in an autologous transfusion program. Phlebotomies were performed at an average gestational age of 32.4 weeks (range 13-40). Changes in mean diastolic blood pressure and pulse were minimal. Electronic fetal monitoring tracings were normal during the 34 procedures in which it was used. The frequency of mild donor reactions (4%) was consistent with that in nonpregnant donors. After entry into this program, 15 patients received a total of 29 U of packed red blood cells (23 autologous; six homologous). Homologous transfusion was avoided in 86.7% of patients receiving blood. Selected pregnant women can participate safely in autologous blood collection programs, minimizing the need, and therefore the risks, of homologous transfusion. PMID:3292974

  16. Autologous gluteal lipograft.

    PubMed

    Nicareta, Beatriz; Pereira, Luiz Haroldo; Sterodimas, Aris; Illouz, Yves Gérard

    2011-04-01

    In the past 25 years, several different techniques of lipoinjection have been developed. The authors performed a prospective study to evaluate the patient satisfaction and the rate of complications after an autologous gluteal lipograft among 351 patients during January 2002 and January 2008. All the patients included in the study requested gluteal augmentation and were candidates for the procedure. Overall satisfaction with body appearance after gluteal fat augmentation was rated on a scale of 1 (poor), 2 (fair), 3 (good), 4 (very good), and 5 (excellent). The evaluation was made at follow-up times of 12 and 24 months. The total amount of clean adipose tissue transplanted to the buttocks varied from 100 to 900 ml. In nine cases, liponecrosis was treated by aspiration with a large-bore needle connected to a 20-ml syringe, performed as an outpatient procedure. Infection of the grafted area also occurred for four patients and was treated by incision drainage and use of antibiotics. Of the 21 patients who expressed the desire of further gluteal augmentation, 16 had one more session of gluteal fat grafting. The remaining five patients did not have enough donor area and instead received gluteal silicone implants. At 12 months, 70% reported that their appearance after gluteal fat augmentation was "very good" to "excellent," and 23% responded that their appearance was "good." Only 7% of the patients thought their appearance was less than good. At 24 months, 66% reported that their appearance after gluteal fat augmentation was "very good" (36%) to "excellent" (30%), and 27% responded that their appearance was "good." However, 7% of the patients continued to think that their appearance was less than good. At this writing, the average follow-up time for this group of patients has been 4.9 years. The key to successful gluteal fat grafting is familiarity with the technique, knowledge of the gluteal topography, and understanding of the patient's goals. With experience, the

  17. Autologous chondrocytes. Autologous chondrocyte implantation: more data needed.

    PubMed

    2011-05-01

    There is no standard surgical treatment for young adults with persistent, incapacitating symptoms of knee cartilage damage. ChondroCelect is the first cell therapy product to be authorised in the European Union. It contains a dense suspension of chondrocytes cultured from a biopsy of the patient's knee cartilage for 4 weeks before being reimplanted. Clinical evaluation of Chondro-Celect only includes one trial, versus subchondral microfracture, in 118 patients. After 3 years of follow-up, there was no difference in the symptom score between the groups. Histological outcome was better after autologous chondrocyte implantation, but methodological problems make it difficult to interpret the observed difference. Long-term functional outcomes remain to be determined. More joint complications occurred after autologous chondrocyte implantation than after subchondral bone microfracture: more frequently symptomatic cartilage hypertrophy (27% versus 13%, possibly related to the implantation technique), joint swelling (22% versus 6.6%), joint effusion (24% versus 9.8%), and joint crepitations (18% versus 6.6%). Autologous chondrocyte implantation was sometimes associated with flu-like syndrome (in 7.8% of patients), which did not occur with the microfracture technique. Autologous chondrocyte implantation is more complex than microfracture. During routine use, there is a risk that one patient will inadvertently receive chondrocytes collected from another patient, leading to a risk of rejection. In practice, this autologous chondrocyte product should only be used by highly specialised teams, and its assessment must continue. PMID:21648176

  18. 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.

  19. Transient transfection of purified Babesia bovis merozoites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II...

  20. Rebooting autoimmunity with autologous HSCT.

    PubMed

    Snowden, John A

    2016-01-01

    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. PMID:26744435

  1. Transfer printing of transfected cell microarrays from poly(ethylene glycol)-oleyl surfaces onto biological hydrogels.

    PubMed

    Yamaguchi, Satoshi; Komiya, Senori; Matsunuma, Erika; Yamahira, Shinya; Kihara, Takanori; Miyake, Jun; Nagamune, Teruyuki

    2013-12-01

    We have developed a novel technique for constructing microarrays of transfected mammalian cells on or in extracellular matrix (ECM) hydrogels by transfer printing from patterned poly(ethylene glycol) (PEG)-oleyl surfaces. A mixed solution of small interfering RNA (siRNA) and a transfection reagent was spotted on PEG-oleyl-coated glass slides using an ink-jet printer, and the cells were then transiently immobilized on the patterned transfection mixtures. After overlaying an ECM hydrogel sheet onto the immobilized cells, the cells sandwiched between the glass slide and the hydrogel sheet were incubated at 37°C for simultaneous transfection of siRNA into cells and adhesion of cells to the hydrogel sheet. Transfer of the adhered, transfected cells was completed by peeling off the hydrogel sheet. The knockdown of a model gene in the transferred cell microarray by the transfected siRNA was successfully confirmed. Transfected cell microarrays were also embedded within three-dimensional ECM hydrogels. In the three-dimensional hydrogel, the inhibition effect of siRNA on cancer cell invasion was evaluated by quantifying the size of cell clusters on the microarrays. These results indicate that transfection of cell microarrays on or in a biological matrix is a promising technique for high-throughput screening of disease-related genes by direct observation of cellular phenomena in a physiologically relevant environment. PMID:23893595

  2. Sendai F/HN Viroplexes for Efficient Transfection of Leukemic T Cells

    PubMed Central

    Kim, Jung Seok; Lee, Yeon Kyung; Jeong, Hwa Yeon; Kang, Seong Jae; Kim, Min Woo; Ryu, Seung Hyun; Kim, Hong Sung; Kim, Keun Sik; Kim, Dong-Eun

    2013-01-01

    Purpose Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. Materials and Methods The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a ζ-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. Results The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. Conclusion This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines. PMID:23918564

  3. Gold nanoparticle mediated laser transfection for high-throughput antisense applications

    NASA Astrophysics Data System (ADS)

    Kalies, S.; Heinemann, D.; Schomaker, M.; Birr, T.; Ripken, T.; Meyer, H.

    2013-06-01

    The delivery of antisense structures, like siRNA, is beneficial for new therapeutic approaches in regenerative sciences. Optical transfection techniques enable high spatial control combined with minimal invasive treatment of cells due to the use of short laser pulses. However, single cell laser transfection by a tightly focused laser beam, for example femtosecond laser transfection, has the major drawback of low throughput. Compared to this, high-throughput in laser transfection is possible by applying gold nanoparticles irradiated by a weakly focused laser beam scanning over the cell sample. Herein, we show the delivery of antisense molecules and demonstrate the minimal cytotoxicity of a method called gold nanoparticle mediated (GNOME) laser transfection. A 532 nm microchip laser in conjugation with 200 nm gold nanoparticles at a concentration of 0.5 μg/cm2 is used. In addition to antisense molecules, the uptake of dextrans of several sizes is analyzed.

  4. Transfection in the third dimension

    PubMed Central

    Dhaliwal, Anandika; Oshita, Victor; Segura, Tatiana

    2013-01-01

    An understanding of parameters that modulate gene transfer in 3-D will assist in the formation of gene delivery systems and scaffolds, which can mediate efficient non-viral delivery for guiding in-vivo tissue regeneration and therapy. We have previously demonstrated the cell area and length, integrin expression, and RhoGTPases mediated signalling to be pivotal parameters that guide gene transfer to mouse mesenchymal stem cells (mMSCs) cultured in 2-D and are modulated by ECM proteins. In this study, we were interested in determining if cationic polymer mediated gene transfer to cells seeded in 3-D would occur through different mechanisms as compared to 2-D. In particular, we examined the endocytosis pathways used to internalize polyplexes, and the role of cytoskeletal dynamics and RhoGTPases on non-viral gene transfer for cells seeded in 2-D and 3-D. Inhibition of clathrin- and caveolae- mediated endocytosis resulted in more drastic decrease in overall transgene expression for cells seeded in 3-D than those in 2-D. In addition, polyplex internalization was only significantly decreased in 3-D when clathrin-mediated endocytosis was inhibited, while caveolae-mediated endocytosis inhibition for cells seeded in 2-D resulted in the strongest polyplex internalization inhibition. Actin and microtubule polymerization affected 2-D and 3-D transfection differently. Microtubule depolymerization enhanced transgene expression in 2-D, but inhibited transgene expression in 3-D. Last, inhibition of RhoGTPases also affected 2-D and 3-D transfection differently. The inhibition of ROCK effector resulted in a decrease of transgene expression and internalization for cells seeded in 3-D, but not 2-D and the inhibition of effector PAK1 resulted in an increase of transgene expression for both 2-D and 3-D. Overall, our study suggests that the process of gene transfer occurs through different mechanisms for cells seeded in 2-D compared to those seeded in 3-D. PMID:23929354

  5. 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. PMID:27471645

  6. 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.

  7. Generation and Cryopreservation of Clinical Grade Wilms' Tumor 1 mRNA-Loaded Dendritic Cell Vaccines for Cancer Immunotherapy.

    PubMed

    Smits, Evelien L J M; Stein, Barbara; Nijs, Griet; Lion, Eva; Van Tendeloo, Viggo F; Willemen, Yannick; Anguille, Sébastien; Berneman, Zwi N

    2016-01-01

    First described in the 1970s, dendritic cells (DC) are currently subjects of intense investigation to exploit their unique antigen-presenting and immunoregulatory capacities. In cancer, DC show promise to elicit or amplify immune responses directed against cancer cells by activating natural killer (NK) cells and tumor antigen-specific T cells. Wilms' tumor 1 (WT1) protein is a tumor-associated antigen that is expressed in a majority of cancer types and has been designated as an antigen of major interest to be targeted in clinical cancer immunotherapy trials. In this chapter, we describe the generation, cryopreservation, and thawing of clinical grade autologous monocyte-derived DC vaccines that are loaded with WT1 by messenger RNA (mRNA) electroporation. This in-house-developed transfection method gives rise to presentation of multiple antigen epitopes and can be used for all patients without restriction of human leukocyte antigen (HLA) type. PMID:27033213

  8. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been widely done using labor intensive and cytotoxic liposome-based transfection reagents....

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  10. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    PubMed

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture. PMID:27259918

  11. [Autologous Fat Grafting in Scar Revision].

    PubMed

    Yu, Pan-xi; Cai, Jing-long

    2016-04-01

    Regenerative medicine is an emerging discipline. Adipose tissue is a rich source of fat cells and mesenchymal stem cells, and autologous fat grafting has increasingly been applied in plastic surgeries and dermatological treatments. This paper reviews the latest advances in autologous fat grafting in scar revision. PMID:27181904

  12. 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

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

    PubMed

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

    2010-07-01

    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

  14. 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

  15. Lipothioureas as Lipids for Gene Transfection: A Review

    PubMed Central

    Breton, Marie; Leblond, Jeanne; Tranchant, Isabelle; Scherman, Daniel; Bessodes, Michel; Herscovici, Jean; Mignet, Nathalie

    2011-01-01

    Non-viral gene therapy requires innovative strategies to achieve higher transfection efficacy. A few years ago, our group proposed bioinspired lipids whose interaction with DNA was not based on ionic interactions, but on hydrogen bonds. We thus developed lipids bearing a thiourea head which allowed an interaction with DNA phosphates through hydrogen bonds. After a proof of concept with a lipid bearing three thiourea functions, a molecular and cellular screening was performed by varying all parts of the lipids: the hydrophobic anchor, the spacer, the linker, and the thiourea head. Two lipothiourea-based structures were identified as highly efficient in vitro transfecting agents. The lipothioureas were shown to reduce non specific interactions with cell membranes and deliver their DNA content intracellularly more efficiently, as compared to cationic lipoplexes. These lipids could deliver siRNA efficiently and allowed specific cell targeting in vitro. In vivo, thiourea lipoplexes presented a longer retention time in the blood and less accumulation in the lungs after an intravenous injection in mice. They also induced luciferase gene expression in muscle and tumor after local administration in mice. Therefore, these novel lipoplexes represent an excellent alternative to cationic lipoplexes as transfecting agents. In this review we will focus on the structure activity studies that permitted the identification of the two most efficient thiourea lipids.

  16. 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.

  17. 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. PMID:27260108

  18. 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

  19. Improved biolistic transfection of hair cells.

    PubMed

    Zhao, Hongyu; Avenarius, Matthew R; Gillespie, Peter G

    2012-01-01

    Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells. PMID:23049715

  20. Improved Biolistic Transfection of Hair Cells

    PubMed Central

    Gillespie, Peter G.

    2012-01-01

    Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca2+-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells. PMID:23049715

  1. 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.

  2. Combining nebulization-mediated transfection and polymer microarrays for the rapid determination of optimal transfection substrates.

    PubMed

    Unciti-Broceta, Asier; Díaz-Mochón, Juan J; Mizomoto, Hitoshi; Bradley, Mark

    2008-01-01

    In this manuscript, we report how transfection efficiencies vary as a function of the substrate upon which cells adhere using a polymer microarray platform to allow rapid analysis of a large number of substrates. During these studies, traditional transfection protocols were nonsatisfactory, and thus we developed an approach in which an ultrasonic nebulizer was used to dispense lipoplexes onto cell-based microarrays in the absence of liquid. Under these conditions, droplets were directly deposited onto the cells thereby enhancing transfection. This approach was successfully applied to the transfection of various cell lines immobilized on a library of polyacrylates and permitted the identification of highly efficient transfection/polymer combinations, while showing that specific polymer-cell interactions may promote the efficacy of chemical transfection. PMID:18247582

  3. Nucleofection as an efficient nonviral transfection method for human monocytic cells.

    PubMed

    Martinet, Wim; Schrijvers, Dorien M; Kockx, Mark M

    2003-07-01

    Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2-6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells. PMID:12889809

  4. MATra - Magnet Assisted Transfection: combining nanotechnology and magnetic forces to improve intracellular delivery of nucleic acids.

    PubMed

    Bertram, J

    2006-08-01

    Recent efforts combining nanotechnology and magnetic properties resulted in the development and commercialization of magnetic nanoparticles that can be used as carriers for nucleic acids for in vitro transfection and for gene therapy approaches including DNA-based vaccination strategies. The efficiency of intracellular delivery is still a limiting factor for basic cell biological research and also for emerging technologies such as temporary gene silencing based on inhibitory RNA/siRNA. Nanotechnology has resulted in a variety of different nanostructures and especially nanoparticles as carriers in a wide range of new drug delivery systems for conventional drugs, recombinant proteins, vaccines and more recently nucleic acids. It is possible to combine superparamagnetic nanoparticles with magnetic forces to increase, direct and optimize intracellular delivery of biomolecules. This article discusses the main approaches in the field of magnet assisted transfection (MATra) focusing on the transfection or intracellular delivery of nucleic acids, although also suitable to improve the intracellular delivery of other biomolecules. PMID:16918404

  5. Sepsis after autologous fat grafting.

    PubMed

    Talbot, Simon G; Parrett, Brian M; Yaremchuk, Michael J

    2010-10-01

    Autologous fat grafting is an increasingly popular technique, with numerous examples of excellent results. Adherence to key principles, including sterile technique and low-volume injection throughout layers of tissue, appears to be critical to obtaining good results. Reports of adverse outcomes are infrequent, but several case reports document both infectious and aesthetic complications. This case report represents an extreme complication, including abscess formation, life-threatening sepsis, and residual deformity. It serves as yet another reminder that early adoption of surgical procedures by those without a sound understanding of the underlying principles and techniques can have disastrous consequences. Furthermore, physicians operating on any patient must understand the potential for complications and be able to manage these appropriately when they occur. PMID:20885205

  6. 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

  7. Nanoparticles of compacted DNA transfect postmitotic cells.

    PubMed

    Liu, Ge; Li, DeShan; Pasumarthy, Murali K; Kowalczyk, Tomasz H; Gedeon, Christopher R; Hyatt, Susannah L; Payne, Jennifer M; Miller, Timothy J; Brunovskis, Peter; Fink, Tamara L; Muhammad, Osman; Moen, Robert C; Hanson, Richard W; Cooper, Mark J

    2003-08-29

    Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore. PMID:12807905

  8. Verification system for postoperative autologous blood retransfusion.

    PubMed

    Yoshikawa, Takeki; Kimura, Eizen; Kobayashi, Shinji; Ishihara, Ken

    2013-01-01

    Medical staff members should match blood products with patients using a barcode authentication system for blood transfusion to prevent medical accidents. However, our hospital only verifies the blood products of the Japanese Red Cross Society and the preserved autologous blood, not the autologous blood salvaged during the operation or from the oxygenator. In this study, we developed the barcode medication administration system and mobile device for verification. This system will prevent blood transfusion errors in the ward setting. PMID:23920751

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

    PubMed Central

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

    2015-01-01

    Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo. PMID:26243452

  10. [Autologous transfusion in obstetrics and fetal safety].

    PubMed

    Rech, F; Patella, A; Cecchi, A; Ippolito, M; Indraccolo, S R

    1994-06-01

    It is common knowledge that for modern medicine transfusion therapy represents a precious resource and an often mandatory option. It is equally known that autohemotransfusion (or autologous transfusion) provides further advantages: certainty of blood availability when necessary, absence of transfusion reactions, elimination of the risk of infections that is still associated with the traditional homologous transfusions. In its most widespread application, autotransfusion provides for the donation of one or more units of autologous blood, mostly before elective surgery. Even in obstetrics the practice of autologous blood donation with the aim of autotransfusion is finding increasing employment. However, there are still controversial aspects and the need is pointed out for more authoritative verifications as refers to the alleged innocuity to the fetus of acute maternal blood loss. The present study was performed to contribute personal experience to a better definition of the possible interactions between autologous blood donation during pregnancy and unborn child welfare. To this end, 80 term pregnant women underwent fetal heart rate electronic monitoring before, during and after the donation of one unit of autologous blood. Both during and after the phlebotomy there were no cardiotocographic signs of fetal hypo-oxygenation. Even the non stress tests performed at a distance of 24 hours and those that were periodically repeated afterwards were normal, confirming the safety of autologous predonation during pregnancy. However, the authors think that in obstetrics it is still premature to consider the experimental phase of autotransfusion as definitively exhausted. PMID:7936387

  11. Optimizacion of Babesia bovis transfection methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Postoperative Autologous Reinfusion in Total Knee Replacement

    PubMed Central

    Crescibene, A.; Martire, F.; Gigliotti, P.; Rende, A.; Candela, M.

    2015-01-01

    Surgeries for total knee replacement (TKR) are increasing and in this context there is a need to develop new protocols for management and use of blood transfusion therapy. Autologous blood reduces the need for allogeneic blood transfusion and the aim of the present study was to verify the safety and the clinical efficacy. An observational retrospective study has been conducted on 124 patients, undergoing cemented total knee prosthesis replacement. Observed population was stratified into two groups: the first group received reinfusion of autologous blood collected in the postoperative surgery and the second group did not receive autologous blood reinfusion. Analysis of data shows that patients undergoing autologous blood reinfusion received less homologous blood bags (10.6% versus 30%; p = 0.08) and reduced days of hospitalization (7.88 ± 0.7 days versus 8.96 ± 2.47 days for the control group; p = 0.03). Microbiological tests were negative in all postoperatively salvaged and reinfused units. Our results emphasize the effectiveness of this procedure and have the characteristics of simplicity, low cost (€97.53 versus €103.79; p < 0.01), and easy reproducibility. Use of autologous drainage system postoperatively is a procedure that allows reducing transfusion of homologous blood bags in patients undergoing TKR. PMID:26442168

  13. Paradoxical regulation of dopamine receptors in transfected 293 cells.

    PubMed

    Filtz, T M; Artymyshyn, R P; Guan, W; Molinoff, P B

    1993-08-01

    , haloperidol, clozapine, and epidepride, alone or in combination with NPA. Incubation of cells with SCH-23390 had no effect on the density of D2 receptors, and SCH-23390 did not block the effect of NPA. D2-selective antagonists increased the density of receptors. D2L receptor mRNA levels were unchanged during agonist treatment. This suggests a role for translational or post-translational mechanisms in the regulation of D2 receptor levels in transfected cell lines. PMID:8102783

  14. Nucleic acid transfection and transgenesis in parasitic nematodes.

    PubMed

    Lok, James B

    2012-04-01

    Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins. PMID:21880161

  15. 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

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

    PubMed

    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

  17. 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

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

    PubMed

    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

  19. 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.

  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. 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

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

    PubMed

    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

  3. 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.

  4. Sweet Syndrome After Autologous Stem Cell Transplant.

    PubMed

    Alkan, Ali; İdemen, Celal; Okçu Heper, Aylin; Utkan, Güngör

    2016-02-01

    Sweet syndrome (acute febrile neutrophilic dermatosis) is a rare clinical entity characterized by skin lesions, neutrophilia, fever, and neutrophilic infiltration of the dermis. It may be a consequence of malignant disease, comorbidities, or drugs. We present a case of acute febrile neutrophilic dermatosis in a patient after autologous stem cell transplant. PMID:25748978

  5. 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

  6. 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. PMID:26779485

  7. 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.

  8. Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

    PubMed

    Obregón-Barboza, Verónica; Del Rincón-Castro, Ma Cristina; Cabrera-Ponce, José L; Ibarra, Jorge E

    2007-03-01

    The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells. PMID:17184851

  9. 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.

  10. Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Hamann, L; Nickel, R; Tannich, E

    1995-01-01

    To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568055

  11. Autologous Fat Grafting Improves Facial Nerve Function

    PubMed Central

    Klinger, Marco; Lisa, Andrea; Caviggioli, Fabio; Maione, Luca; Murolo, Matteo; Vinci, Valeriano; Klinger, Francesco Maria

    2015-01-01

    We describe the case of a 45-year-old male patient who presented a retractile and painful scar in the nasolabial fold due to trauma which determined partial motor impairment of the mouth movements. We subsequently treated him with autologous fat grafting according to Coleman's technique. Clinical assessments were performed at 5 and 14 days and 1, 3, and 6 months after surgical procedure and we observed a progressive release of scar retraction together with an important improvement of pain symptoms. A second procedure was performed 6 months after the previous one. We observed total restoration of mimic movements within one-year follow-up. The case described confirms autologous fat grafting regenerative effect on scar tissue enlightening a possible therapeutic effect on peripheral nerve activity, hypothesizing that its entrapment into scar tissue can determine a partial loss of function. PMID:26167327

  12. Achieving ideal breast aesthetics with autologous reconstruction

    PubMed Central

    2015-01-01

    Achieving ideal breast aesthetic has become a top priority for women considering breast reconstruction following mastectomy. The use of autologous tissue is generally regarded as providing the most natural results because donor tissues quality and consistency is similar to that of the native breast. There are several donor sites that are particularly useful for autologous reconstruction that include the abdomen, gluteal region, posterior thorax, and the thigh. Traditional and microsurgical techniques can be used. Shaping is a critical component and involves a basic understanding of the footprint, conus, and skin envelope. This manuscript will review many aspects of breast shaping in-order to achieve aesthetically pleasing results in a predictable manner. PMID:26005645

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

    PubMed Central

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

    2016-01-01

    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 tumor neovascularization. PMID:27356780

  14. 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. PMID:24750320

  15. Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line.

    PubMed

    Fernández Fernández, Elena; Santos-Carballal, Beatriz; Weber, Wolf-Michael; Goycoolea, Francisco M

    2016-04-11

    Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw ∼ 20 kDa and degree of acetylation ∼ 30%). The average diameter of positively-charged complexes (i.e. ζ ∼+25 mV) was ∼ 200 nm. The complexes were found relatively stable over 14h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF. PMID:26875537

  16. 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

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

    PubMed

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

    2016-01-01

    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

  18. 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

  19. 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

    2016-01-01

    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

  20. Expression of transfected mutant beta-actin genes: transitions toward the stable tumorigenic state.

    PubMed Central

    Leavitt, J; Ng, S Y; Varma, M; Latter, G; Burbeck, S; Gunning, P; Kedes, L

    1987-01-01

    Mutant human beta-actin genes were introduced into normal human (KD) fibroblasts and the derivative cell line HuT-12, which is immortalized but nontumorigenic, to test their ability to promote conversion to the tumorigenic state. Transfected substrains of HuT-12 fibroblasts that expressed abundant levels of mutant beta-actin (Gly-244----Asp-244) produced subcutaneous tumors in athymic mice after long latent periods (1.5 to 3 months). However, transfected substrains of KD fibroblasts retained their normal finite life span in culture and consequently were incapable of producing tumors. Substrains of HuT-12 cells transfected with the wild-type beta-actin gene and some transfected strains that expressed low or undetectable levels of mutant beta-actin did not produce tumors. Cell lines derived from transfectant cell tumors always exhibited elevated synthesis of the mutant beta-actin, ranging from 145 to 476% of the level expressed by the transfected cells that were inoculated to form the tumor. In general, primary transfectant cells that expressed the highest levels of mutant beta-actin were more tumorigenic than strains that expressed lower levels. The tumor-derived strains were stable in tumorigenicity and produced tumors with shortened latent periods of only 2 to 4 weeks. These findings imply that the primary transfectant strains develop subpopulations of cells that are selected to form tumors because of their elevated rate of exogenous mutant beta-actin synthesis. Actin synthesis and accumulation of gamma-actin mRNA from the endogenous beta- and gamma-actin genes were diminished in tumor-derived strains, apparently to compensate for elevated mutant beta-actin synthesis and maintain the normal cellular concentration of actin. Synthesis of the transformation-sensitive tropomyosin isoforms was decreased along with mutant beta-actin expression. Such modulations in tropomyosin synthesis are characteristically seen in transformation of avian, rodent, and human fibroblasts

  1. [Nutritional pathway for autologous stem cell transplantation].

    PubMed

    Aoyama, Takashi; Imataki, Osamu; Inoue, Naomi; Katsumata, Mina; Katsuta, Tomoko; Kataoka, Tomomi; Yoshida, Takashi; Mochizuki, Takahiro; Motokawa, Satoshi; Tamai, Yotaro; Hagiwara, Shotaro; Kawakami, Kimihiro

    2007-08-01

    We developed a nutritional pathway for autologous stem cell transplantation (SCT) to be applied in our transplantation unit. We performed autologous SCT for 37 patients with malignant lymphoma and multiple myeloma during from April 2003 to July 2005. For 10 of them who underwent SCT since 2005,we intervened with nutritional support using our original nutritional pathway,to monitor the clinical course of SCT from the aspect of dietetics with a dietician making assessments of the individual nutrition status. From comparing the 2 groups with (n=27) or without (n=10) the nutritional pathway, oral intake at day 14 was significantly increased from 1,038 kcal to 1,440 kcal,and at discharge developed from 1,167 kcal to 1,446 kcal without statistical significance. Patients whose body weight decreased more than 5% were reduced from 52%(14/27) to 10%(1/10),and 3 days reduction of the CVC insertion period was observed after the intervention. Although the long-term clinical outcome was not fully evaluated, the efficacy of nutritional pathway for autologous SCT was suggested. PMID:17687206

  2. Exogenous hTERT gene transfected endothelial progenitor cells from bone marrow promoted angiogenesis in ischemic myocardium of rats

    PubMed Central

    Li, Shang-Hai; Wang, Dan-Dan; Xu, Yun-Jun; Ma, Guo-Dong; Li, Xing-Yue; Liang, Wei-Jun

    2015-01-01

    Objective: To explore the biological behavior and the revascularizative ability of endothelial progenitor cells (EPCs) transfected with human telomerase reverse transcriptase (hTERT) gene. Methods: EPCs were isolated from mononuclear cells in bone marrow by using the method of density gradient centrifugation, then cultured with differential velocity adherent method, EPCs were transfected by recombinant plasmid carrying GFP report gene EGFP-hTERT. The EPCs secretion and proliferation ability were detected before and after transfection. The expression of EPCs mRNA were detected by RT-PCR before and after transfection. The new capillaries of infarct area were observed. Results: After transgenesis, the proliferation of EPCs were increased, and the secretion of NO, LDH, iNOS by EPCs were significantly increased compared to the non-transgenesis group. After transplanted the transfected EPCs into the ischemic myocardial of rats, revascularization were increased obviously. Conclusion: EPCs maintained the original biological characteristics after transfecting exogenous hTER gene, the proliferation and survival rate were up-regulated significantly, and the revascularization ability of EPCs were significantly strengthen. PMID:26550433

  3. Gene transfection by echo contrast agent microbubbles

    NASA Astrophysics Data System (ADS)

    Tachibana, Katsuro

    2002-11-01

    In vitro and in vivo experiments have demonstrated that various echo contrast agent microbubbles can be intentionally ruptured by diagnostic and therapeutic ultrasound. Violent microstreaming are produced during microbubble collapse. Researchers have hypothesized that these microjets or microstreaming could be applied to promote diffusion of drugs into various tissues and lesions. The most exciting application of this method is probably delivery of genes into cells. As various genes are currently under investigation for the purpose of treating diseases, ultrasound and microbubbles may be used as a modality to promote better outcome for gene therapy. Recent studies have shown that different gases contained within the bubbles greatly influence the degree of gene transfection. Also, the outer layer of the microbubbles can be custom-made for binding to target tissue. Recent advance on this topic will be discussed.

  4. 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.

  5. 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. PMID:24131074

  6. Single cell transfection using plasmid decorated AFM probes

    SciTech Connect

    Cuerrier, Charles M.; Lebel, Rejean; Grandbois, Michel . E-mail: michel.grandbois@usherbrooke.ca

    2007-04-13

    Eukaryotic cells were individually transfected using commercially available atomic force microscope tips decorated with plasmidic DNA encoding for the fluorescent protein EGFP. In a typical transfection attempt, the tip is forcibly incorporated into the cell thus allowing for the transfer of the genetic material through the cell membrane. A sharp discontinuity, corresponding to the passage of the tip through the cell membrane can be easily detected when monitoring the cellular deformation as a function of the applied force. In order for the transfection to be successful, the tip must reversibly penetrates the membrane without causing disturbance or damage to the cell. Transfection success rate (30%), cell survival, and growth are confirmed by epifluorescence microscopy. This technique provides an alternative tool to the transfection toolbox, allowing the transfection of specific individual cells with minimal disturbance.

  7. Phenotypic MicroRNA Microarrays

    PubMed Central

    Kwon, Yong-Jun; Heo, Jin Yeong; Kim, Hi Chul; Kim, Jin Yeop; Liuzzi, Michel; Soloveva, Veronica

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

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

    PubMed

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

    2015-08-01

    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

  9. Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection.

    PubMed

    Anada, Takahisa; Karinaga, Ryouji; Koumoto, Kazuya; Mizu, Masami; Nagasaki, Takeshi; Kato, Yoshio; Taira, Kazunari; Shinkai, Seiji; Sakurai, Kazuo

    2005-11-28

    Our previous work showed that a natural beta-(1-->3)-d-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)(80) on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system. PMID:16219384

  10. Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.

    PubMed

    Raghupathy, Mohan B; Griffiths, Jonathan S; Stobbs, Lorne W; Brown, Daniel C W; Brandle, James E; Wang, Aiming

    2006-09-01

    The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection. PMID:16777241

  11. Quantitative study of effects of free cationic chains on gene transfection in different intracellular stages.

    PubMed

    Cai, Jinge; Yue, Yanan; Wang, Yanjing; Jin, Zhenyu; Jin, Fan; Wu, Chi

    2016-09-28

    Previously, we revealed that in the application of using cationic polymer chains, polyethylenimine (PEI), to condense anionic plasmid DNA chains (pDNA) to form the DNA/polymer polyplexes, after all the pDNAs are complexed with PEI, further added PEIs exist individual chains and free in the solution mixture. It is those uncomplexed polycation chains that dramatically promote the gene transfection. In the current study, we studied how those free cationic chains with different lengths and topologies affect the intracellular trafficking of the polyplexes, the translocation of pDNA through the nuclear membrane, the transcription of pDNA to mRNA and the translocation of mRNA from nucleus to cytosol in HepG2 cells by using a combination of the three-dimensional confocal microscope and TaqMan real-time PCR. We found that free branched PEI chains with a molar mass of 25,000g/mol and a total concentration of 1.8×10(-6)g/mL promote the overall gene transfection efficiency by a factor of ~500 times. Our results quantitatively reveal that free chains help little in the cellular uptake, but clearly reduce the lysosomal entrapment of those internalized polyplexes (2-3 folds); assist the translocation of pDNA through nuclear membrane after it is released from the polyplexes in the cytosol (~5 folds); enhance the pDNA-to-mRNA transcription efficiency (~4 folds); and facilitate the nucleus-to-cytosol translocation of mRNA (7-8 folds). The total enhancement of those steps agrees well with the overall efficiency, demonstrating, for the first time, how free cationic polymer chains quantitatively promote the gene transfection in each step in the intracellular space. PMID:27448443

  12. 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.

  13. 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.

  14. Correlation between cationic lipid-based transfection and cell division.

    PubMed

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. PMID:25556666

  15. Unexpected transcellular protein crossover occurs during canonical DNA transfection.

    PubMed

    Arsenault, Jason; Cuijpers, Sabine A G; Niranjan, Dhevahi; Davletov, Bazbek

    2014-12-01

    Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection. PMID:25043607

  16. Autologous Graft-versus-Tumor Effect: Reality or Fiction?

    PubMed Central

    2016-01-01

    In contrast to allogeneic hematopoietic stem cell transplantation, the current dogma is not an evidence of graft-versus-tumor effect in autologous hematopoietic stem cell transplantation; thus, it is assumed that autologous hematopoietic stem cell transplantation only relies on the high-dose chemotherapy to improve clinical outcomes. However, recent studies argue in favor of the existence of an autologous graft-versus-tumor without the detrimental complications of graft-versus-host disease due to the nonspecific immune response from the infused donor alloreactive immune effector cells in allogeneic hematopoietic stem cell transplantation. Herein, this paper reviews the clinical evidence of an autologous graft-versus-tumor effect based on the autograft collected and infused host immune effector cells and host immunity recovery after autologous hematopoietic stem cell transplantation affecting clinical outcomes in cancer patients.

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

    PubMed

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

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. PMID:25957917

  18. Breast Augmentation With Autologous Fat Injection

    PubMed Central

    Li, Fa-Cheng; Chen, Bing; Cheng, Lin

    2014-01-01

    Introduction Autologous fat transplantation has attracted great interest in breast augmentation for cosmetic purpose. In the present study, we reported our experience in fat grafting in breast in 105 cases, and some detailed procedure concerning efficacy and safety of grafting was evaluated. Methods Fat was harvested using 20-mL syringe attached to a 3-hole blunt cannula in a diameter not beyond 3 mm. After washing with cool normal saline to remove blood, the fat was managed with open method using cotton towel as a platform for concentration fat tissue and separating them from fluids, oil, and debris. A 14-gauge, 1-hole blunt cannula was used to place the fat through 3-mm incision on inframammary fold. The fat was infiltrated into the breast from deep to superficial subcutaneous plane. Results Between July 2002 and August 2010, 105 patients have undergone this procedure. The age distribution of the patients ranged from 18 to 45 years, with a mean of 31.3 years. Grafted fat volume has ranged from 120 to 250 mL (average, 205 mL) per breast per session. All women had a significant improvement in their breast size and shape postoperatively, and the breasts were soft and natural in appearance. Conclusions Liposuction and autologous fat transplantation is a suitable approach for augmentation mammaplasty. PMID:25003461

  19. Autologous stem cells for personalised medicine.

    PubMed

    Prasongchean, Weerapong; Ferretti, Patrizia

    2012-09-15

    Increasing understanding of stem cell biology, the ability to reprogramme differentiated cells to a pluripotent state and evidence of multipotency in certain adult somatic stem cells has opened the door to exciting therapeutic advances as well as a great deal of regulatory and ethical issues. Benefits will come from the possibility of modelling human diseases and develop individualised therapies, and from their use in transplantation and bioengineering. The use of autologous stem cells is highly desirable, as it avoids the problem of tissue rejection, and also reduces ethical and regulatory issues. Identification of the most appropriate cell sources for different potential applications, development of appropriate clinical grade methodologies and large scale well controlled clinical trials will be essential to assess safety and value of cell based therapies, which have been generating much hope, but are by and large not yet close to becoming standard clinical practice. We briefly discuss stem cells in the context of tissue repair and regenerative medicine, with a focus on individualised clinical approaches, and give examples of sources of autologous cells with potential for clinical intervention. PMID:22561284

  20. Water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride as a nucleic acids vector for cell transfection.

    PubMed

    Faizuloev, Evgeny; Marova, Anna; Nikonova, Alexandra; Volkova, Irina; Gorshkova, Marina; Izumrudov, Vladimir

    2012-08-01

    To endow the cationic polysaccharides with solubility in the whole pH-range without loss of functionality of the amino groups, different chitosan samples were treated with glycidyltrimethylammonium chloride. Each modified unit of the exhaustively alkylated quaternized chitosan (QCht) contained both quaternary and secondary amino groups. The intercalated dye displacement assay and ζ-potential measurements implied stability of QCht polyplexes at physiological conditions and protonation of the secondary amino groups in slightly acidic media which is favorable for transfection according to proton sponge mechanism. The cytotoxicity and transfection efficacy increased with the chain lengthening. Nevertheless, the longest chains of QCht, 250 kDa were less toxic than PEI for COS-1 cells and revealed comparable and even significantly higher transfection activity of siRNA and plasmid DNA, respectively. Thus, highly polymerized QCht (250 kDa) provided the highest level of the plasmid DNA transfection being 5 and 80 times more active than QCht (100 kDa) and QCht (50 kDa), respectively, and 4-fold more effective than PEI, 25 kDa. The established influence of QCht molecular weight on toxicity and transfection efficacy allows elaborating polysaccharide vectors that possess rational balance of these characteristics. PMID:24750918

  1. Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

    PubMed Central

    Krauss, R D; Bubien, J K; Drumm, M L; Zheng, T; Peiper, S C; Collins, F S; Kirk, K L; Frizzell, R A; Rado, T A

    1992-01-01

    We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR. Images PMID:1372253

  2. EGFP-EGF1-Conjugated PLGA Nanoparticles for Targeted Delivery of siRNA into Injured Brain Microvascular Endothelial Cells for Efficient RNA Interference

    PubMed Central

    Chen, Chen; Mei, Heng; Shi, Wei; Deng, Jun; Zhang, Bo; Guo, Tao; Wang, Huafang; Hu, Yu

    2013-01-01

    Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer. PMID:23593330

  3. 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. PMID:23262422

  4. 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

  5. Pressure-Mediated Oligonucleotide Transfection of Rat and Human Cardiovascular Tissues

    NASA Astrophysics Data System (ADS)

    Mann, Michael J.; Gibbons, Gary H.; Hutchinson, Howard; Poston, Robert S.; Hoyt, E. Grant; Robbins, Robert C.; Dzau, Victor J.

    1999-05-01

    The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

  6. Twin disulfides as opportunity for improving stability and transfection efficiency of oligoaminoethane polyplexes.

    PubMed

    Klein, Philipp M; Müller, Katharina; Gutmann, Christina; Kos, Petra; Krhac Levacic, Ana; Edinger, Daniel; Höhn, Miriam; Leroux, Jean-Christophe; Gauthier, Marc A; Wagner, Ernst

    2015-05-10

    The synthesis of precise gene delivery vehicles by solid-supported chemistry is an effective way to establish structure-activity relationships and optimize existing transfection carriers. Sequence-defined cationic oligomers with different topologies were modified with twin disulfide-forming cysteine-arginine-cysteine (CRC) motifs. The influence of this motif versus single disulfide on the biophysical properties and biological performance of polyplexes was investigated, with pDNA and siRNA as nucleic acid cargoes. Clear differences between structures with isolated cysteines and CRC motifs were observed with respect to properties like nucleic acid binding, serum stability, response to reducing agents, and gene transfer/silencing. The main observed effect of the CRC motif was to increase polyplex stability. The consequences for nucleic acid delivery were less predictable and depended on oligomer topology. For some oligomers intrinsically forming stable polyplexes (i.e., already in the absence of CRC motif), this further stabilization resulted in a reduction or even loss in transfection efficiency. For PEGylated and targeted oligomers with intrinsically less stable polyplex structures, this modification led to a significant enhancement in transfection efficiency. PMID:25553827

  7. The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA.

    PubMed Central

    Perales, B; Ortín, J

    1997-01-01

    The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA. PMID:8995663

  8. An analysis of a preoperative pediatric autologous blood donation program

    PubMed Central

    Letts, Merv; Perng, Richard; Luke, Brian; Jarvis, James; Lawton, Louis; Hoey, Steve

    2000-01-01

    Objective To determine the efficacy of a pediatric autologous blood donation program. Design A retrospective study of patient charts and blood-bank records. Setting The Children’s Hospital of Eastern Ontario, Ottawa, a tertiary care, pediatric centre. Patients One hundred and seventy-three children who received blood transfusions for a total of 182 procedures between June 1987 and June 1997. Interventions Autologous and homologous blood transfusion required for major surgical intervention, primarily spinal fusion. Main outcome measures Surgeons’ accuracy in predicting the number of autologous blood units required for a given procedure, compliance rate (children’s ability to donate the requested volume of blood), utilization rate of autologous units and rate of allogeneic transfusion. Results The surgeons’ accuracy in predicting the number of autologous units required for a given procedure was 53.8%. The compliance rate of children to donate the requested amount of blood was 80.3%. In children below the standard age and weight criteria for blood donation the compliance rate was 75.5%. The utilization rate of autologous units obtained was 84.4% and the incidence of allogeneic transfusion was 26.6%. Conclusions There was a high rate of compliance and utilization of predonated autologous blood in the children in the study. Preoperative blood donation programs are safe and effective in children, even in those below the standard age and weight criteria of 10 years and 40 kg. PMID:10812347

  9. Role of autologous bladder-neck slings: a urogynecology perspective.

    PubMed

    Zoorob, Dani; Karram, Mickey

    2012-08-01

    The concept of the autologous pubovaginal sling involves supporting the proximal urethra and bladder neck with a piece of graft material, achieving continence either by providing a direct compressive force on the urethra/bladder outlet or by reestablishing a reinforcing platform or hammock against which the urethra is compressed during transmission of increased abdominal pressure. Pubovaginal slings using a biological sling material (whether autologous, allograft, or xenograft) can be used successfully to manage primary or recurrent stress incontinence. This article addresses the indications for the use of an autologous bladder-neck sling, describes the surgical techniques, and discusses outcomes and technical considerations. PMID:22877713

  10. Indium-111 autologous leukocyte imaging in pancreatitis

    SciTech Connect

    Anderson, J.R.; Spence, R.A.; Laird, J.D.; Ferguson, W.R.; Kennedy, T.L.

    1986-03-01

    Thirty-nine patients with acute pancreatitis have been assessed using a prognostic factor grading system, abdominal ultrasound, and autologous leukocyte imaging. Both prognostic factor grading and leukocyte imaging can accurately assess the severity of the disease early in its course. All patients with a negative indium-labeled leukocyte image recovered without sequelae, whereas five of the 12 patients with a positive image developed complications, including two deaths. Abdominal ultrasound is of no value in assessing severity, but is a useful method of detecting those patients with gallstone-associated disease. In patients with suspected abscess formation following acute pancreatitis, indium leukocyte imaging does not differentiate between fat necrosis and abscess formation. In this situation, computerized tomography should be carried out before laparotomy is undertaken.

  11. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    EPA Science Inventory

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  12. Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line.

    PubMed

    Binder, R; MacDonald, C C; Burch, J B; Lazier, C B; Williams, D L

    1990-02-01

    A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2330000

  13. Autologous and allogeneic serum eye drops. The Dutch perspective.

    PubMed

    van der Meer, Pieter F; Seghatchian, Jerard; de Korte, Dirk

    2015-08-01

    If regular artificial tears are ineffective for treatment of ocular surface disorders (including extreme dry eye syndrome), serum eye drops (SEDs) may provide a way to relieve the symptoms. However, not all patients are eligible to donate blood to produce autologous SEDs. Therefore, the use of allogeneic SEDs (obtained from voluntary blood donors) should be explored as an alternative for autologous SEDs. The Dutch blood bank organization is currently looking into the possibilities to provide allogeneic SEDs, as (GMP) regulations become stricter, making it for hospitals more difficult to provide autologous SEDs. To demonstrate effectiveness of both autologous and allogeneic SEDs, a clinical trial is planned. The current status of SEDs in The Netherlands is described. This paper is based on summary of the presentation given at the DGTI meeting in Dresden. PMID:26138910

  14. 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. PMID:26590947

  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. PMID:26260195

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. In vitro gene transfection using dendritic poly(L-lysine).

    PubMed

    Ohsaki, Mio; Okuda, Tatsuya; Wada, Akihiro; Hirayama, Toshiya; Niidome, Takuro; Aoyagi, Haruhiko

    2002-01-01

    Monodispersed dendritic poly(L-lysine)s (DPKs) of several generations were synthesized, and their characteristics as a gene transfection reagent were then investigated. The agarose gel shift and ethidium bromide titration assay proved that the DPKs of the third generation and higher could form a complex with a plasmid DNA, and the degree of compaction of the DNA was increased by the increasing number of the generation. The DPKs of the fifth and sixth generation, which have 64 and 128 amine groups on the surface of the molecule, respectively, showed efficient gene transfection ability into several cultivated cell lines without significant cytotoxity. In addition, the transfection efficiency of the DPK of the sixth generation was not seriously reduced even if serum was added at 50% of the final concentration into the transfection medium. Because we can strictly synthesize various DPK derivatives, which have several types of branch units, terminal cationic groups, and so on, they are expected to be a good object of study regarding the basic information on the detailed mechanism of gene transfection into cells. We also expect to be able to easily construct DPK-based functional gene carriers, e.g., DPKs modified by ligands such as a sugar chain, which can enable advanced gene delivery in vivo. PMID:12009940

  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. PMID:10726885

  2. Improved assays for xenosensor activation based on reverse transfection.

    PubMed

    Küblbeck, Jenni; Anttila, Teemu; Pulkkinen, Juha T; Honkakoski, Paavo

    2015-10-01

    Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z' factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies. PMID:26187274

  3. 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. PMID:17025362

  4. Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

    PubMed Central

    Prinsen, C F; Veerkamp, J H

    1998-01-01

    We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect. PMID:9425108

  5. Enhanced pyrimidine dimer repair in cultured murine epithelial cells transfected with the denV gene of bacteriophage T4.

    PubMed

    Kusewitt, D F; Budge, C L; Ley, R D

    1994-04-01

    The patch size for excision repair of ultraviolet radiation (UV)-induced pyrimidine dimers was determined in cultured murine epithelial cells with normal and enhanced pyrimidine dimer repair capabilities. Cells with enhanced pyrimidine dimer repair were produced by transfecting 308 cells with the denV gene of bacteriophage T4; this gene encodes the enzyme endonuclease V. Pyrimidine dimer repair following exposure to UV from an FS-40 sunlamp was determined by micrococcal dimer-specific nuclease digestion and alkaline sucrose ultracentrifugation. Patch size ws estimated based on the photolytic lability of bromodeoxyuridine-substituted DNA. Excision repair of UV-induced pyrimidine dimers in denV-transfected 308 cells was enhanced two- to threefold. Production of mRNA from the denV gene in cell lines with enhanced repair was confirmed by RNA blotting. In control cells, the patch size for excision repair of DNA photoproducts was estimated to be 34 nucleotides per photoproduct removed; in denV-transfected cells, a smaller average patch size of 10-16 nucleotides per photoproduct removed was calculated. Thus, endonuclease V activity appears to alter not only the extent, but also the nature of excision repair in UV-exposed mammalian epithelial cells. PMID:8151125

  6. Second autologous stem cell transplant for multiply relapsed Hodgkin's disease.

    PubMed

    Lin, T S; Avalos, B R; Penza, S L; Marcucci, G; Elder, P J; Copelan, E A

    2002-05-01

    Therapeutic options for patients with Hodgkin's disease who relapse after high-dose chemotherapy with autologous stem cell support are limited. Salvage chemotherapy is not curative, and allogeneic stem cell transplantation in this setting is associated with mortality rates of 40-65%. We report our institution's experience with second autologous transplants in this patient population. Five patients (median age 36) with relapsed Hodgkin's disease underwent a second autologous stem cell transplant at a median of 66 months after first transplant. Four patients received CBV, and one patient received BuCy as conditioning. Neutrophil and platelet engraftment occurred by days +10 and +16, respectively. All patients achieved a complete response, and no relapses have occurred after a median follow-up of 42 months. All four patients who received CBV developed interstitial pneumonitis, and two patients died of pulmonary complications 37 and 48 months following second transplant. Three patients remain alive and disease-free 41, 42 and 155 months after second transplant. These data indicate that second autologous transplantation should be considered for selected patients who relapse after a prolonged response to first autologous transplant. However, BCNU pneumonitis is the major toxicity in patients who have undergone previous mantle radiation and received busulfan with first transplant. PMID:12040474

  7. 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.

  8. Chimeric autologous/allogeneic constructs for skin regeneration.

    PubMed

    Rasmussen, Cathy Ann; Tam, Joshua; Steiglitz, Barry M; Bauer, Rebecca L; Peters, Noel R; Wang, Ying; Anderson, R Rox; Allen-Hoffmann, B Lynn

    2014-08-01

    The ideal treatment for severe cutaneous injuries would eliminate the need for autografts and promote fully functional, aesthetically pleasing autologous skin regeneration. NIKS progenitor cell-based skin tissues have been developed to promote healing by providing barrier function and delivering wound healing factors. Independently, a device has recently been created to "copy" skin by harvesting full-thickness microscopic tissue columns (MTCs) in lieu of autografts traditionally harvested as sheets. We evaluated the feasibility of combining these two technologies by embedding MTCs in NIKS-based skin tissues to generate chimeric autologous/allogeneic constructs. Chimeric constructs have the potential to provide immediate wound coverage, eliminate painful donor site wounds, and promote restoration of a pigmented skin tissue possessing hair follicles, sweat glands, and sebaceous glands. After MTC insertion, chimeric constructs and controls were reintroduced into air-interface culture and maintained in vitro for several weeks. Tissue viability, proliferative capacity, and morphology were evaluated after long-term culture. Our results confirmed successful MTC insertion and integration, and demonstrated the feasibility of generating chimeric autologous/allogeneic constructs that preserved the viability, proliferative capacity, and structure of autologous pigmented skin. These feasibility studies established the proof-of-principle necessary to further develop chimeric autologous/allogeneic constructs for the treatment of complex skin defects. PMID:25102552

  9. Autologous splenic transplantation for splenic trauma.

    PubMed Central

    Pisters, P W; Pachter, H L

    1994-01-01

    OBJECTIVE: The authors reviewed the experimental evidence, surgical technique, complications, and results of clinical trials evaluating the role of autologous splenic transplantation for splenic trauma. SUMMARY BACKGROUND DATA: Splenorrhaphy and nonoperative management of splenic injuries have now become routine aspects in the management of splenic trauma. Unfortunately, not all splenic injuries are readily amenable to conventional spleen-conserving approaches. Heterotopic splenic autotransplantation has been advocated for patients with severe grade IV and V injuries that would otherwise mandate splenectomy. For this subset of patients, splenic salvage by autotransplantation would theoretically preserve the critical role the spleen plays in the host's defense against infection. METHODS: The relevant literature relating to experimental or clinical aspects of splenic autotransplantation was identified and reviewed. Data are presented on the experimental evaluation of autogenous splenic transplantation, methods and complications of autotransplantation, choice of anatomic site and autograft size, and results of clinical trials in humans. RESULTS: The most commonly used technique of autotransplantation in humans involves implanting tissue homogenates or sections of splenic parenchyma into pouches created in the gastrocolic omentum. Most authors have observed evidence of splenic function with normalization of postsplenectomy thrombocytosis, immunoglobulin M levels, and peripheral blood smears. Some degree of immune function of transplanted grafts has been demonstrated with in vivo assays, but the full extent of immunoprotection provided by human splenic autotransplants is currently unknown. CONCLUSIONS: Multiple human and animal studies have established that splenic autotransplantation is a relatively safe and easily performed procedure that results in the return of some hematologic and immunologic parameters to baseline levels. Some aspects of reticuloendothelial

  10. Slug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma

    PubMed Central

    2011-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract. We assessed the relevance of Slug in measuring the invasive potential of ESCC cells in vitro and in vivo in immunodeficient mice. Methods We utilized RNA interference to knockdown Slug gene expression, and effects on survival and invasive carcinoma were evaluated using a Boyden chamber transwell assay in vitro. We evaluated the effect of Slug siRNA-transfection and Slug cDNA-transfection on E-cadherin and Bcl-2 expression in ESCC cells. A pseudometastatic model of ESCC in immunodeficient mice was used to assess the effects of Slug siRNA transfection on tumor metastasis development. Results The EC109 cell line was transfected with Slug-siRNA to knockdown Slug expression. The TE13 cell line was transfected with Slug-cDNA to increase Slug expression. EC109 and TE13 cell lines were tested for the expression of apoptosis-related genes bcl-2 and metastasis-related gene E-cadherin identified previously as Slug targets. Bcl-2 expression was increased and E-cadherin was decreased in Slug siRNA-transfected EC109 cells. Bcl-2 expression was increased and E-cadherin was decreased in Slug cDNA-transfected TE13 cells. Invasion of Slug siRNA-transfected EC109 cells was reduced and apoptosis was increased whereas invasion was greater in Slug cDNA-transfected cells. Animals injected with Slug siRNA-transfected EC109 cells exhihited fewer seeded nodes and demonstrated more apoptosis. Conclusions Slug down-regulation promotes cell apoptosis and decreases invasion capability in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of metastatic ESCC. PMID:21599940

  11. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  12. [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. PMID:27349114

  13. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. PMID:24863237

  14. Multiplexing nano-electroporation for simultaneous transfection of multiple cells

    NASA Astrophysics Data System (ADS)

    Howdyshell, M.; Vieira, G.; Gallego-Perez, D.; Zhao, X.; Lee, L. J.; Sooryakumar, R.

    2013-03-01

    Transfection of biomolecules into cells via electrophoresis across nanochannels, or nano-electroporation, is a recently developed technique shown to deliver precisely controlled dosages with low cell mortality rates. Such advantages are due to the nanochannels used for transfection, which distinguish this technique from bulk and micro-electroporation. Recent demonstrations of nano-electroporation rely on optical tweezers for cell localization, which restrict throughput to sequential electroporation of one cell at a time. In the current work, we overcome this drawback by advancing a multiplexed approach that integrates the nano-channel device with an array of magnetic traps remotely controlled by external magnetic fields. This setup enables multiple magnetically labeled cells to be manipulated in parallel, allowing for simultaneous electroporation of many cells with precisely controlled dosages. After transfection, the cells can be moved downstream for further analysis. Such a magnetically-actuated, remotely-controlled approach for loading of cells and subsequent removal of transfected cells has the potential to transform the current device into an automated platform for simultaneous dosage-controlled biomolecule delivery to large numbers of individual cells.

  15. Autologous blood donation in support of cardiac surgery: a preliminary report on a hospital-based autologous donor programme.

    PubMed

    Pinkerton, P H

    1994-11-01

    The purpose of this study was to assess the success or otherwise of the introduction of an autologous blood programme in support of cardiac surgery in reducing patient exposure to allogeneic blood products and to assess the guideline of two units as the collection schedule for such patients. Sixty-six patients were enrolled in the programme provided they met defined clinical conditions and donated one, two or three units of blood at seven-day intervals, using isovolaemic conditions. One minor vasovagal adverse reaction was recorded. Of the 66 patients, 51 (77%) avoided allogeneic red cells and 42 (64%) received no allogeneic product. If each patient deposited two units, 51 (77%) would have required no allogeneic red cells; if three units were deposited, 57 (86%) patients would have required no allogeneic red cells, but 60 units would be surplus to requirements. Comparison of 52 patients for coronary artery bypass grafting who were autologous donors, with 130 patients undergoing the procedure before the availability of autologous blood, supports the suggestions that there is increased readiness to initiate transfusion of autologous blood and that exposure to allogeneic red cells is reduced. However, exposure to allogeneic products of all kinds is not reduced. It is concluded that the collection of two units of autologous blood is appropriate for most eligible patients and that this reduces exposure to allogeneic red cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7828248

  16. Autologous blood injection for the treatment of recurrent mandibular dislocation.

    PubMed

    Coser, R; da Silveira, H; Medeiros, P; Ritto, F G

    2015-08-01

    The purpose of the present study was to evaluate the effectiveness of autologous blood injection in the treatment of recurrent temporomandibular joint dislocation. Eleven patients diagnosed with recurrent dislocation of the joint that could not be self-reduced, received bilateral injections of autologous blood in the superior joint compartment and pericapsular region. During a follow-up period ranging from 24 to 35 months (average 29.6 months), eight patients (72.7%) did not show new episodes of dislocation. The most advocated treatment for recurrent dislocation is eminectomy, which involves a skin incision, with the risk of damaging the facial nerve, requires general anaesthesia, and presents an average success rate of 85% according to the literature. Autologous blood injection is a simple, rapid, minimally invasive, and cost-effective technique, with a low possibility of complications, and is a feasible alternative treatment before surgical intervention. PMID:26022511

  17. Improving diagnosis of appendicitis. Early autologous leukocyte scanning

    SciTech Connect

    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 appendicitis 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-imaging In 111 oxyquinoline autologous leukocyte scanning is a practical and highly accurate adjunct for diagnosing appendicitis.

  18. Therapeutic Potential of Autologous Stem Cell Transplantation for Cerebral Palsy

    PubMed Central

    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. PMID:23259143

  19. Specific transfection of inflamed brain by macrophages: a new therapeutic strategy for neurodegenerative diseases.

    PubMed

    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

  20. Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection.

    PubMed

    Namvar, Ali; Bolhassani, Azam; Khairkhah, Niloofardokht; Motevalli, Fatemeh

    2015-07-01

    Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer-based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra- and extracellular barriers. The recent progress has shown that stimuli-responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA-polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. PMID:25761628

  1. Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.

    PubMed Central

    Liang, T J; Makdisi, W J; Sun, S; Hasegawa, K; Zhang, Y; Wands, J R; Wu, C H; Wu, G Y

    1993-01-01

    A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions. Images PMID:8383700

  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. Localization and functional analysis of CHIP28k water channels in stably transfected Chinese hamster ovary cells.

    PubMed

    Ma, T; Frigeri, A; Tsai, S T; Verbavatz, J M; Verkman, A S

    1993-10-25

    CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proximal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced approximately 8 x 10(6) copies of CHIP28k protein/cell. Plasma membrane osmotic water permeability (Pf), measured by stopped-flow light scattering, was 0.004 cm/s in control (vector-transfected) cells (10 degrees C) and 0.014 cm/s in the CHIP28k-transfected cells. Pf in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgCl2. CHIP28k expression did not affect the transport of protons and the small polar non-electrolytes urea and formamide. CHIP28k immunoreactivity and function was then determined in subcellular fractions. Pf in 6-carboxyfluorescein-labeled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002 cm/s (control cells) and 0.011 cm/s (CHIP28k-transfected cells); Pf in transfected cells was inhibited by HgCl2. Immunoblotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (approximately 5000 monomers/microns 2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies establish a stably transfected somatic cell line that strongly expresses functional CHIP28k water channels. As in the original proximal tubule cells

  4. Techniques of autologous pericardial leaflet replacement for aortic valve reconstruction.

    PubMed

    Rankin, J Scott; Nöbauer, Christian; Crooke, Philip S; Schreiber, Christian; Lange, Rüdiger; Mazzitelli, Domenico

    2014-08-01

    Glutaraldehyde-fixed autologous pericardium rarely calcifies or retracts, and it is a useful substitute for cardiac valve leaflets. Current understanding of aortic valve geometry provides good models for aortic leaflet design, and pericardial leaflet construction is illustrated in this article for bicuspid and tri-leaflet valves. Outcomes have been characterized by low valve-related complication rates, and results of recent series are encouraging. Perhaps sufficient data are available to consider autologous pericardial leaflet replacement in highly selected younger patients with irreparable leaflets and contraindications to warfarin. PMID:25087813

  5. A review of the application of autologous blood transfusion.

    PubMed

    Zhou, J

    2016-01-01

    Autologous blood transfusion (ABT) has been gradually attracting more attention due to the increasingly prominent problem of blood transfusion safety and blood shortage in recent years. With the rapid development of blood conservation techniques, blood component separation technology, blood transfusion medicine and a constant increase in clinical needs, ABT technology has been expanded and innovated to a large degree. In this study, the development of preoperative autologous blood donation (PABD), acute normovolemic hemodilution (ANH), intraoperative and postoperative autotransfusion, and other new technologies and theories are reviewed and existing questions are analyzed. Challenges and applications are also discussed in order to provide reference for peers. PMID:27533770

  6. Autologous hematopoietic stem cell transplantation for mediastinal extramedullary plasmocytoma.

    PubMed

    Abdelkefi, Abderrahmene; Ben Othman, Tarek; Torjman, Lamia; Ladeb, Saloua; Ben Ghorbel, Imed; Lakhal, Amed; Ben Amor, Ramzi; Miled, Mohamed; Kchir, Mohamed-Nidhameddine; Ben Abdeladhim, Abdeladhim

    2003-07-01

    Extramedullary plasmocytoma (EMP) is a rare cell neoplasm most frequently localised in the upper respiratory tract. We report the case of a 43 year-old-man, with an unusual presentation of EMP developing in the mediastinum, two years after a diagnosis of solitary plasmocytoma of the bone which was successfully treated by local irradiation. In this aggressive presentation, we decided to perform an autologous hematopoietic stem cell transplantation. Two months after transplantation, CT scan showed disappearance of the mediastinal mass and immunofixation of the serum was normal. Selected cases of diffuse EMP, could benefit from intensive treatment followed by autologous hematopoietic stem cell transplantation. PMID:14534964

  7. A review of the application of autologous blood transfusion

    PubMed Central

    Zhou, J.

    2016-01-01

    Autologous blood transfusion (ABT) has been gradually attracting more attention due to the increasingly prominent problem of blood transfusion safety and blood shortage in recent years. With the rapid development of blood conservation techniques, blood component separation technology, blood transfusion medicine and a constant increase in clinical needs, ABT technology has been expanded and innovated to a large degree. In this study, the development of preoperative autologous blood donation (PABD), acute normovolemic hemodilution (ANH), intraoperative and postoperative autotransfusion, and other new technologies and theories are reviewed and existing questions are analyzed. Challenges and applications are also discussed in order to provide reference for peers. PMID:27533770

  8. Autologous bone-marrow mesenchymal cell induced chondrogenesis (MCIC).

    PubMed

    Huh, Sung Woo; Shetty, Asode Ananthram; Ahmed, Saif; Lee, Dong Hwan; Kim, Seok Jung

    2016-01-01

    Degenerative and traumatic articular cartilage defects are common, difficult to treat, and progressive lesions that cause significant morbidity in the general population. There have been multiple approaches to treat such lesions, including arthroscopic debridement, microfracture, multiple drilling, osteochondral transplantation and autologous chondrocyte implantation (ACI) that are currently being used in clinical practice. Autologous bone-marrow mesenchymal cell induced chondrogenesis (MCIC) is a single-staged arthroscopic procedure. This method combines a modified microfracture technique with the application of a bone marrow aspirate concentrate (BMAC), hyaluronic acid and fibrin gel to treat articular cartilage defects. We reviewed the current literatures and surgical techniques for mesenchymal cell induced chondrogenesis. PMID:27489409

  9. 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

  10. 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 ...

  11. 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

  12. Intraoperative Indocyanine Green Laser Angiography in Pediatric Autologous Ear Reconstruction.

    PubMed

    Martins, Deborah B; Farias-Eisner, Gina; Mandelbaum, Rachel S; Hoang, Han; Bradley, James P; Lee, Justine C

    2016-05-01

    Skin flap vascularity is a critical determinant of aesthetic results in autologous ear reconstruction. In this study, we investigate the use of intraoperative laser-assisted indocyanine green angiography (ICGA) as an adjunctive measure of skin flap vascularity in pediatric autologous ear reconstruction. Twenty-one consecutive pediatric patients undergoing first-stage autologous total ear reconstruction were retrospectively evaluated. The first 10 patients were treated traditionally (non-ICGA), and the latter 11 patients were evaluated with ICGA intraoperatively after implantation of the cartilage construct and administration of suction. Relative and absolute perfusion units in the form of contour maps were generated. Statistical analyses were performed using independent sample Student t test. Statistically significant differences in exposure and infection were not found between the 2 groups. However, decreased numbers of surgical revisions were required in cases with ICGA versus without ICGA (P = 0.03), suggesting that greater certainty in skin flap perfusion correlated with a reduction in revision surgeries. In cases of exposure, we found an average lowest absolute perfusion unit of 14.3, whereas cases without exposure had an average of 26.1 (P = 0.02), thereby defining objective parameters for utilizing ICGA data in tailoring surgical decision making for this special population of patients. Defined quantitative parameters for utilizing ICGA in evaluating skin flap vascularity may be a useful adjunctive technique in pediatric autologous ear reconstruction. PMID:27579233

  13. Potential of autologous NK cell therapy to eradicate leukemia

    PubMed Central

    Abdel-Azim, Hisham; Heisterkamp, Nora

    2015-01-01

    B-precursor acute lymphoblastic leukemia (BP-ALL) patients are immunocompromised. We recently reported that functional natural killer (NK) cells can be grown from patient bone marrow and blood samples at diagnosis. Surprisingly, such NK cells exhibit cytotoxicity against autologous BP-ALL cells. Here, we outline unanswered questions, challenges and possible applications associated with these findings. PMID:25949882

  14. Intraoperative Indocyanine Green Laser Angiography in Pediatric Autologous Ear Reconstruction

    PubMed Central

    Martins, Deborah B.; Farias-Eisner, Gina; Mandelbaum, Rachel S.; Hoang, Han; Bradley, James P.

    2016-01-01

    Summary: Skin flap vascularity is a critical determinant of aesthetic results in autologous ear reconstruction. In this study, we investigate the use of intraoperative laser-assisted indocyanine green angiography (ICGA) as an adjunctive measure of skin flap vascularity in pediatric autologous ear reconstruction. Twenty-one consecutive pediatric patients undergoing first-stage autologous total ear reconstruction were retrospectively evaluated. The first 10 patients were treated traditionally (non-ICGA), and the latter 11 patients were evaluated with ICGA intraoperatively after implantation of the cartilage construct and administration of suction. Relative and absolute perfusion units in the form of contour maps were generated. Statistical analyses were performed using independent sample Student t test. Statistically significant differences in exposure and infection were not found between the 2 groups. However, decreased numbers of surgical revisions were required in cases with ICGA versus without ICGA (P = 0.03), suggesting that greater certainty in skin flap perfusion correlated with a reduction in revision surgeries. In cases of exposure, we found an average lowest absolute perfusion unit of 14.3, whereas cases without exposure had an average of 26.1 (P = 0.02), thereby defining objective parameters for utilizing ICGA data in tailoring surgical decision making for this special population of patients. Defined quantitative parameters for utilizing ICGA in evaluating skin flap vascularity may be a useful adjunctive technique in pediatric autologous ear reconstruction. PMID:27579233

  15. 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.

  16. 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

  17. Ultrasonic enhancement of gene transfection in murine melanoma tumors.

    PubMed

    Miller, D L; Bao, S; Gies, R A; Thrall, B D

    1999-11-01

    The enhancement of gene transfection by ultrasound (US) was evaluated in vitro and in vivo using the B16 mouse melanoma model. Cultured cells were either exposed in suspensions in vitro or implanted subcutaneously in female C57BL/6 mice for 10-14 days and, subsequently exposed, in vivo. For comparison to results with a luciferase plasmid, a reporter plasmid for green fluorescent protein (GFP) was used to evaluate transfection efficiency. US was supplied by a system, similar to a Dornier HM-3 lithotripter, that produced shock waves (SW) of 24.4 MPa peak positive and 5.2 MPa peak negative pressure amplitudes at the focus. The plasmids were mixed with the suspensions to achieve 20 ,microL mL(-1), or were injected intratumorally to provide 0.2 mg DNA per mL of tumor. Acoustic cavitation was promoted by retaining 0.2 mL of air in the 1.2-mL exposure chambers in vitro and by injecting air at 10% of tumor volume in vivo. In vitro, cell counts declined to 5.3% of shams after 800 SW exposure, with 1.4% of the cells expressing GFP after 2 days of culture. In vivo, 2 days after 400 SW exposure, viable-cell recovery from excised tumors was reduced to 4.2% of shams and cell transfection was enhanced by a factor of about 8, reaching 2.5% of cell counts (p < 0.005 in t-test). These results show that strong tumor ablation induced by US shock wave treatment can be coupled with simultaneous enhancement of gene transfection. PMID:10626630

  18. Isolation and Transfection of Primary Culture Bovine Retinal Pericytes.

    PubMed

    Primo, Vincent A; Arboleda-Velasquez, Joseph F

    2016-01-01

    This protocol describes an enzymatic approach for isolating homogeneous cultures of pericytes from retinas of bovine source. In summary, retinas are dissected, washed, digested, filtered, cultured in specific media to select for pericytes, and finally expanded for a low passage culture of about 14 million bovine retinal pericytes (BRP) within 4-6 weeks. This protocol also describes a liposomal-based technique for transfection of BRPs. PMID:27172949

  19. RNA topology

    PubMed Central

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases. PMID:23603781

  20. 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.

  1. Cell transfection as a tool to study growth hormone action

    SciTech Connect

    Norstedt, G.; Enberg, B.; Francis, S.

    1994-12-31

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determined pathways whereby GH signals are conveyed within the cell. 33 refs., 2 tabs.

  2. DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure.

    PubMed

    Liu, Ying; Yan, Jing; Santangelo, Philip J; Prausnitz, Mark R

    2016-07-28

    Ultrasound has been studied as a promising tool for intracellular gene delivery. In this work, we studied gene transfection of a human prostate cancer cell line exposed to megahertz pulsed ultrasound in the presence of contrast agent and assessed the efficiency of fluorescently labelled DNA delivery into cell nuclei, which is necessary for gene transfection. At the sonication conditions studied, ~30% of cells showed DNA uptake 30min after sonication, but that fraction decreased over time to ~10% of cells after 24h. Most cells containing DNA had DNA in their nuclei, but the amount varied significantly. Transfection efficiency peaked at ~10% at 8h post sonication. Among those cells containing DNA, ~30% of DNA was localized in the cell nuclei, ~30% was in autophagosomes/autophagolysosomes and the remainder was "free" in the cytoplasm 30min after sonication. At later times up to 24h, ~30% of DNA continued to be found in the nuclei and most or all of the rest of the DNA was in autophagosomes/autophagolysosomes. These results demonstrate that ultrasound can deliver DNA into cell nuclei shortly after sonication and that the rest of the DNA can be cleared by autophagosomes/autophagolysosomes. PMID:27165808

  3. 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.

  4. Laser-based patterning for transfected cell microarrays.

    PubMed

    Hook, Andrew L; Creasey, Rhiannon; Hayes, Jason P; Thissen, Helmut; Voelcker, Nicolas H

    2009-12-01

    The spatial control over biomolecule- and cell-surface interactions is of great interest to a broad range of biomedical applications, including sensors, implantable devices and cell microarrays. Microarrays in particular require precise spatial control and the formation of patterns with microscale features. Here, we have developed an approach specifically designed for transfected cell microarray (TCM) applications that allows microscale spatial control over the location of both DNA and cells on highly doped p-type silicon substrates. This was achieved by surface modification, involving plasma polymerization of allylamine, grafting of poly(ethylene glycol) and subsequent excimer laser ablation. DNA could be delivered in a spatially defined manner using ink-jet printing. In addition, electroporation was investigated as an approach to transfect attached cells with adsorbed DNA and good transfection efficiencies of approximately 20% were observed. The ability of the microstructured surfaces to spatially direct both DNA adsorption and cell attachment was demonstrated in a functional TCM, making this system an exciting platform for chip-based functional genomics. PMID:20811112

  5. Impact of plasmid quality on lipoplex-mediated transfection.

    PubMed

    De La Vega, Jonathan; Braak, Bas Ter; Azzoni, Adriano R; Monteiro, Gabriel A; Prazeres, Duarte Miguel F

    2013-11-01

    This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials. PMID:23996350

  6. 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. PMID:24863227

  7. Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for Efficient Gene Expression

    PubMed Central

    Kim, Hyejin; Park, Yongkuk; Lee, Jong Bum

    2015-01-01

    Although mRNA has several advantages over plasmid DNA when delivered into cells for gene expression, mRNA transfection is a very rare occurrence in gene delivery. This is mainly because of the labile nature of RNA, resulting in a low expression level of the desired protein. In this study, self-assembled mRNA nanoparticles (mRNA-NPs) packed with multiple repeats of mRNA were synthesized to achieve efficient gene expression. This approach required only a one-step process to synthesize particles with a minimal amount of plasmid DNA to produce the RNA transcripts via rolling circle transcription. Moreover, there are no concerns for cytotoxicity which can be caused by chemical condensates because mRNA-NPs are made entirely of mRNA. An examination of the cells transfected with the mRNA-NPs encoding the green fluorescence protein (GFP) confirmed that the mRNA-NPs can be used as a novel platform for effective gene delivery. PMID:26235529

  8. 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

  9. 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

  10. The effect of the suspension cells in plasma gene transfection method

    NASA Astrophysics Data System (ADS)

    Isozaki, Yuki; Nakano, Koki; Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Tachibana, Kunihide; Jinno, Masafumi

    2015-09-01

    Plasma gene transfection method is a unique technique for introducing nucleic acids into cells by using plasma irradiation. In our previous works, plasma gene transfection method was performed for the adherent cells, e.g. COS-7 cells, and the influence of plasma on gene transfection has been investigated. As a next step for plasma medicine, transfection to much more various kinds of target cells is required. In this study, the authors attempted gene transfection to two kinds of suspension and four kinds of adherent cells. Although the transfection ratios to the suspension cells were low, transfection to all the kinds of cells were validated. To upregulate the transfection ratio for suspension cells, the authors are validating related factors by plasma irradiation. This work was partly supported by JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (Number 25108509, 15H00896) and a grant from Ehime University.

  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. Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA.

    PubMed

    Kwok, Albert; McCarthy, David; Hart, Stephen L; Tagalakis, Aristides D

    2016-05-01

    The effects of lysine peptide lengths on DNA and siRNA packaging and delivery were studied using four linear oligolysine peptides with 8 (K8), 16 (K16), 24 (K24) and 32 (K32) lysines. Oligolysine peptides with 16 lysines or longer were effective for stable monodisperse particle formation and optimal transfection efficiency with plasmid DNA (pDNA), but K8 formulations were less stable under anionic heparin challenge and consequently displayed poor transfection efficiency. However, here we show that the oligolysines were not able to package siRNA to form stable complexes, and consequently, siRNA transfection was unsuccessful. These results indicate that the physical structure and length of cationic peptides and their charge ratios are critical parameters for stable particle formation with pDNA and siRNA and that without packaging, delivery and transfection cannot be achieved. PMID:26684657

  13. Tandem autologous versus autologous/allogeneic transplantation for multiple myeloma: propensity score analysis.

    PubMed

    Kawamura, Koji; Ikeda, Takashi; Hagiwara, Shotaro; Mori, Takehiko; Shinagawa, Atsushi; Nishiwaki, Kaichi; Ohashi, Kazuteru; Kubonishi, Shiro; Fukuda, Takahiro; Ito, Toshiro; Tomita, Naoto; Ichinohe, Tatsuo; Kato, Koji; Morishima, Yasuo; Atsuta, Yoshiko; Sunami, Kazutaka; Kanda, Yoshinobu

    2016-09-01

    Autologous hematopoietic stem cell transplantation (auto-HCT) is considered a standard therapy for transplant-eligible patients with multiple myeloma, while allogeneic HCT (allo-HCT) is controversial. We retrospectively analyzed 765 patients with myeloma who underwent tandem transplantation between 1998 and 2012 using Japanese registry data. We evaluated the clinical outcomes of tandem auto-HCT (n = 676) and auto/allo-HCT (n = 89). To adjust for a selection bias, we compared overall survival (OS) between the two groups by a propensity score analysis. The probability of OS at six years was 58.5% for the tandem auto-HCT group and 54.4% for the tandem auto/allo-HCT group (p = 0.47). In a matched-pair analysis based on the propensity score, the difference in survival between the two groups was not statistically significant, although the survival curve appeared to reach a plateau beyond five years in the auto/allo group. Further strategies to reduce treatment-related mortality and enhance a graft-versus-myeloma effect are necessary to improve OS. PMID:26961137

  14. Tumor site-specific silencing of NF-κB p65 by targeted hollow gold nanospheres-mediated photothermal transfection

    PubMed Central

    Lu, Wei; Zhang, Guodong; Zhang, Rui; Flores, Leo G; Huang, Qian; Gelovani, Juri G; Li, Chun

    2010-01-01

    Nuclear factor-κB (NF-κB) transcription factor is a critical regulator of the expression of genes involved in tumor formation and progression. Successful RNA interference (RNAi) therapeutics targeting NF-κB is challenged by siRNA delivery systems, which can render targeted in vivo delivery, efficient endo-lysosomal escape and dynamic control over activation of RNAi. Here, we report near-infrared light-inducible NF-κB down-regulation through folate receptor-targeted hollow gold nanospheres carrying siRNA recognizing NF-κB p65 subunit. Using micro-positron emission tomography/computed tomography imaging, the targeted nanoconstructs exhibited significantly higher tumor uptake in nude mice-bearing HeLa cervical cancer xenografts than non-targeted nanoparticles following intravenous administration. Mediated by hollow gold nanospheres, controllable cytoplasmic delivery of siRNA was obtained upon near-infrared light irradiation through photothermal effect. Efficient down-regulation of NF-κB p65 was achieved only in tumors irradiated with near-infrared light, but not in non-irradiated tumors grown in the same mice. Liver, spleen, kidney, and lung were not affected by the treatments, in spite of significant uptake of the siRNA nanoparticles in these organs. We term this mode of action “photothermal transfection”. Combined treatments with p65 siRNA photothermal transfection and irinotecan caused substantially enhanced tumor apoptosis and significant tumor growth delay compared with other treatment regimens. Therefore, photothermal transfection of NF-κB p65 siRNA could effectively sensitize the tumor to chemotherapeutic agents. Because NIR light can penetrate skin and be delivered with high spatiotemporal control, therapeutic RNAi may benefit from this novel transfection strategy while avoiding unwanted side effect. PMID:20388791

  15. 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. PMID:25887802

  16. miRNA143 Induces K562 Cell Apoptosis Through Downregulating BCR-ABL

    PubMed Central

    Liang, Bing; Song, Yanbin; Zheng, Wenling; Ma, Wenli

    2016-01-01

    Background Leukemia seriously threats human health and life. MicroRNA regulates cell growth, proliferation, apoptosis, and cell cycle. Whether microRNA could be treated as a target for leukemia is still unclear and the mechanism by which microRNA143 regulates K562 cells needs further investigation. Material/Methods miRNA143 and its scramble miRNA were synthesized and transfected to K562 cells. MTT assay was used to detect K562 cell proliferation. Flow cytometry and a caspase-3 activity detection kit were used to test K562 cell apoptosis. Western blot analysis was performed to determine breakpoint cluster region-Abelson (BCR-ABL) expression. BCR-ABL overexpression and siRNA were used to change BCR-ABL level, and cell apoptosis was detected again after lipofection transfection. Results miRNA143 transfection inhibited K562 cell growth and induced its apoptosis. miRNA143 transfection decreased BCR-ABL expression. BCR-ABL overexpression suppressed miRNA143-induced K562 cell apoptosis, while its reduction enhanced miRNA143-induced apoptosis. Conclusions miRNA143 induced K562 cell apoptosis through downregulating BCR-ABL. miRNA143 might be a target for a new leukemia therapy. PMID:27492780

  17. High transfection efficiency of quantum dot-antisense oligonucleotide nanoparticles in cancer cells through dual-receptor synergistic targeting.

    PubMed

    Zhang, Ming-Zhen; Li, Cheng; Fang, Bi-Yun; Yao, Ming-Hao; Ren, Qiong-Qiong; Zhang, Lin; Zhao, Yuan-Di

    2014-06-27

    Incorporating ligands with nanoparticle-based carriers for specific delivery of therapeutic nucleic acids (such as antisense oligonucleotides and siRNA) to tumor sites is a promising approach in anti-cancer strategies. However, nanoparticle-based carriers remain insufficient in terms of the selectivity and transfection efficiency. In this paper, we designed a dual receptor-targeted QDs gene carrier QD-(AS-ODN+GE11+c(RGDfK)) which could increase the cellular uptake efficiency and further enhance the transfection efficiency. Here, the targeting ligands used were peptides GE11 and c(RGDfK) which could recognize epidermal growth factor receptors (EGFR) and integrin ανβ3 receptors, respectively. Quantitative flow cytometry and ICP/MS showed that the synergistic effect between EGFR and integrin ανβ3 increased the cellular uptake of QDs carriers. The effects of inhibition agents showed the endocytosis pathway of QD-(AS-ODN+GE11+c(RGDfK)) probe was mainly clathrin-mediated. Western blot confirmed that QD-(AS-ODN+GE11+c(RGDfK)) could further enhance gene silencing efficiency compared to QD-(AS-ODN+GE11) and QD-(AS-ODN+c(RGDfK)), suggesting this dual receptor-targeted gene carrier achieved desired transfection efficiency. In this gene delivery system, QDs could not only be used as a gene vehicle but also as fluorescence probe, allowing for localization and tracking during the delivery process. This transport model is very well referenced for non-viral gene carriers to enhance the targeting ability and transfection efficiency. PMID:24896735

  18. 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

  19. 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.

  20. 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

  1. Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells.

    PubMed Central

    Risso, S; DeFelice, L J; Blakely, R D

    1996-01-01

    1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced currents (approximately -100 pA at -60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1-mediated outward current occurs

  2. A nanoparticle formulation that selectively transfects metastatic tumors in mice

    PubMed Central

    Yang, Jian; Hendricks, William; Liu, Guosheng; McCaffery, J. Michael; Kinzler, Kenneth W.; Huso, David L.; Vogelstein, Bert; Zhou, Shibin

    2013-01-01

    Nanoparticle gene therapy holds great promise for the treatment of malignant disease in light of the large number of potent, tumor-specific therapeutic payloads potentially available for delivery. To be effective, gene therapy vehicles must be able to deliver their therapeutic payloads to metastatic lesions after systemic administration. Here we describe nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI. Compared with a state-of-the-art commercially available in vivo gene delivery formulation, i.v. delivery of the core/PEGylated shell (CPS) nanoparticles provided more than a 16,000-fold increase in the ratio of tumor to nontumor transfection. The vast majority of examined liver and lung metastases derived from a colorectal cancer cell line showed transgene expression after i.v. CPS injection in an animal model of metastasis. Histological examination of tissues from transfected mice revealed that the CPS nanoparticles selectively transfected neoplastic cells rather than stromal cells within primary and metastatic tumors. However, only a small fraction of neoplastic cells (<1%) expressed the transgene, and the extent of delivery varied with the tumor cell line, tumor site, and host mouse strain used. Our results demonstrate that these CPS nanoparticles offer substantial advantages over previously described formulations for in vivo nanoparticle gene therapeutics. At the same time, they illustrate that major increases in the effectiveness of such approaches are needed for utility in patients with metastatic cancer. PMID:23959886

  3. 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.

  4. 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

  5. Conceptual and technical aspects of transfection and gene delivery.

    PubMed

    Kaestner, Lars; Scholz, Anke; Lipp, Peter

    2015-03-15

    Genetically modified animals are state of the art in biomedical research as gene therapy is a promising perspective in the attempt to cure hereditary diseases. Both approaches have in common that modified or corrected genetic information must be transferred into cells in general or into particular cell types of an organism. Here we give an overview of established and emerging methods of transfection and gene delivery and provide conceptual and technical advantages and drawbacks of their particular use. Additionally, based on a flow chart, we compiled a rough guideline to choose a gene transfer method for a particular field of application. PMID:25677659

  6. Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro

    PubMed Central

    Xu, Fang-Tian; Li, Hong-Mian; Yin, Qing-Shui; Liang, Zhi-Jie; Huang, Min-Hong; Chi, Guang-Yi; Huang, Lu; Liu, Da-Lie; Nan, Hua

    2015-01-01

    To investigate whether activated autologous platelet-rich plasma (PRP) can promote proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in vitro. hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3rd passage. PRP was collected and activated from human peripheral blood of the same patient. Cultured hASCs were treated with normal osteogenic inductive media alone (group A, control) or osteogenic inductive media plus 5%, 10%, 20%, 40%PRP (group B, C, D, E, respectively). Cell proliferation was assessed by CCK-8 assay. mRNA expression of osteogenic marker genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) and core binding factor alpha 1 (Cbfa1) were determined by Real-Time Quantitative PCR Analysis (qPCR). Data revealed that different concentrations of activated autologous PRP significantly promoted hASCs growth in the proliferation phase compared to the without PRP group and resulted in a dose-response relationship. At 7-d and 14-d time point of the osteogenic induced stage, ALP activity in PRP groups gradually increased with the increasing of concentrations of PRP and showed that dose-response relationship. At 21-d time point of the osteogenic induced stage, PRP groups make much more mineralization and mRNA relative expression of ALP, OPN, OCN and Cbfa1 than that without PRP groups and show that dose-response relationship. This study indicated that different concentrations of activated autologous PRP can promote cell proliferation at earlier stage and promote osteogenic differentiation at later stage of hASCs in vitro. Moreover, it displayed a dose-dependent effect of activated autologous PRP on cell proliferation and osteogenic differentiation of hASCs in vitro. PMID:25901195

  7. 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.

  8. A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo

    PubMed Central

    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. PMID:26728941

  9. Allogeneic and autologous bone marrow transplantation for acute nonlymphocytic leukemia.

    PubMed

    Hurd, D D

    1987-12-01

    Current results show that 50% of young patients with ANLL who undergo allogeneic BMT experience prolonged DFS and may be cured. Encouraging results with high-dose chemo/radiotherapy and autologous BMT are likewise being reported. In addition, some studies using intensive postremission treatment without BMT have shown results comparable to many transplant series. As better ways of preventing GVHD are found, the morbidity and mortality of allogeneic BMT should be reduced and the benefits of transplantation for curing patients with ANLL should be increased. However, the applicability of allogeneic BMT will remain limited due to the availability of compatible donors whether related or unrelated. Further studies are needed in the use of postremission intensive therapy with and without autologous bone marrow support. However, results to date should engender the same degree of enthusiastic optimism that followed the early reports of improved outcome with allogeneic BMT when applied to first remission patients. PMID:3321445

  10. 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

  11. Correction of deep gluteal depression by autologous fat grafting.

    PubMed

    Lewis, C M

    1992-01-01

    In the past, the traditional method of contouring the iliac crest and lateral femoral areas has been liposuction or the surgical removal of the bulges. Unfortunately, this method fails to correct the deep gluteal depression juxtaposed at these two sites. Since we use autologous fat grafts to correct contouring deficiencies elsewhere, it seems logical to investigate whether this technique is applicable to correcting this deformity. We have performed autologous fat grafting to the gluteal depression on 12 patients who underwent lipoplasty of the iliac crest and lateral femoral sites. The longest followup was one year. We have found that this method corrects the deep gluteal depression and yields an improved aesthetic contour. This article describes the technique, addresses the problems encountered, and shows postoperative results. PMID:1626462

  12. Autologous Hematopoietic Stem Cell Transplantation for Multiple Myeloma without Cryopreservation

    PubMed Central

    Al-Anazi, Khalid Ahmed

    2012-01-01

    High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation is considered the standard of care for multiple myeloma patients who are eligible for transplantation. The process of autografting comprises the following steps: control of the primary disease by using a certain induction therapeutic protocol, mobilization of stem cells, collection of mobilized stem cells by apheresis, cryopreservation of the apheresis product, administration of high-dose pretransplant conditioning therapy, and finally infusion of the cryopreserved stem cells after thawing. However, in cancer centers that treat patients with multiple myeloma and have transplantation capabilities but lack or are in the process of acquiring cryopreservation facilities, alternatively noncryopreserved autologous stem cell therapy has been performed with remarkable success as the pretransplant conditioning therapy is usually brief. PMID:22693672

  13. Computer-assisted selection of donor sites for autologous grafts

    NASA Astrophysics Data System (ADS)

    Krol, Zdzislaw; Zeilhofer, Hans-Florian U.; Sader, Robert; Hoffmann, Karl-Heinz; Gerhardt, Paul; Horch, Hans-Henning

    1997-05-01

    A new method is proposed for a precise planning of autologous bone grafts in cranio- and maxillofacial surgery. In patients with defects of the facial skeleton, autologous bone transplants can be harvested from various donor sites in the body. The preselection of a donor site depends i.a. on the morphological fit of the available bone mass and the shape of the part that is to be transplanted. A thorough planning and simulation of the surgical intervention based on 3D CT studies leads to a geometrical description and the volumetric characterization of the bone part to be resected and transplanted. Both, an optimal fit and a minimal lesion of the donor site are guidelines in this process. We use surface similarity and voxel similarity measures in order to select the optimal donor region for an individually designed transplant.

  14. 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

  15. Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

    PubMed Central

    Woods, Georgia; Zito, Karen

    2008-01-01

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices. PMID:19066564

  16. Improvement of efficiency and viability in plasma gene transfection by plasma minimization and optimization electrode configuration

    NASA Astrophysics Data System (ADS)

    Jinno, Masafumi; Tachibana, Kunihide; Motomura, Hideki; Saeki, Noboru; Satoh, Susumu

    2016-07-01

    Plasma gene transfection is expected as a safe and useful method of gene transfection. However, in this method, there is difficulty in keeping both high transfection efficiency and less cell damage simultaneously. The authors have evaluated transfection efficiency and cell viability using four different plasma sources, such as arc discharge, plasma jet, dielectric barrier discharge (DBD), and microplasma. A high transfection efficiency was achieved by discharge forms in which the electric current flows via the cells. This suggested that an electric current plays an important role in plasma gene transfection. The total volume of gas flow must be small or zero and the area in which the cells are directly irradiated by plasma must be small in order to achieve a higher cell viability. The microplasma that satisfies these conditions achieved both the highest transfection efficiency and the highest cell viability simultaneously.

  17. Incomplete defect filling after third generation autologous chondrocyte implantation

    PubMed Central

    Pietschmann, Matthias F.; Ficklscherer, Andreas; Gülecyüz, Mehmet F.; Hammerschmid, Florian; Müller, Peter E.

    2016-01-01

    Introduction Third generation autologous chondrocyte implantation (ACI) is a suitable method for the treatment of cartilage defects in the knee joint. However, knowledge about the development of graft thickness and the clinical relevance of incomplete defect filling in the postoperative course is low. This prospective study analyses the graft integration into the surrounding cartilage, with special consideration of the graft thickness. Material and methods A total of 71 consecutive patients with 79 cartilage defects were treated with third generation autologous chondrocyte implantation (NOVOCART 3D) in the knee. Follow-up magnetic resonance imaging (MRI) was performed at 0.25, 0.5, 1 and 2 years. Graft thickness was measured compared to the surrounding healthy cartilage. The International Knee Documentation Committee (IKDC) scoring system and the visual analogue scale (VAS) were used for clinical evaluation. Cartilage defect filling was classified as the percentage of the surrounding cartilage. Results The average graft thickness showed a significant increase between 3 and 6 months after autologous chondrocyte implantation. Incomplete defect filling occurred in 44 (55.7%) cases. Of these, 33 cases showed incomplete defect filling grade I (> 75%), 10 cases were grade II (> 50%) and one case grade III (> 25%). Incomplete defect filling grade IV (< 25%) was not observed. Incomplete defect filling occurred significantly more often in women (p = 0.021), without worse clinical results. Conclusions Graft thickness after third generation autologous chondrocyte implantation shows increasing graft thickness over the period of 2 years postoperatively. A high rate of incomplete defect filling in the surrounding cartilage was observed, without worse clinical results. PMID:27478460

  18. 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. PMID:26126219

  19. 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.

  20. 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. PMID:26741268

  1. 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

  2. Delayed Cranioplasty: Outcomes Using Frozen Autologous Bone Flaps.

    PubMed

    Hng, Daniel; Bhaskar, Ivan; Khan, Mumtaz; Budgeon, Charley; Damodaran, Omprakash; Knuckey, Neville; Lee, Gabriel

    2015-09-01

    Reconstruction of skull defects following decompressive craniectomy is associated with a high rate of complications. Implantation of autologous cryopreserved bone has been associated with infection rates of up to 33%, resulting in considerable patient morbidity. Predisposing factors for infection and other complications are poorly understood. Patients undergoing cranioplasty between 1999 and 2009 were identified from a prospectively maintained database. Records and imaging were reviewed retrospectively. Demographics, the initial craniectomy and subsequent cranioplasty surgeries, complications, and outcomes were recorded. A total of 187 patients underwent delayed cranioplasty using autologous bone flaps cryopreserved at -30°C following decompressive craniectomy. Indications for craniectomy were trauma (77.0%), stroke (16.0%), subarachnoid hemorrhage (2.67%), tumor (2.14%), and infection (2.14%). There were 64 complications overall (34.2%), the most common being infection (11.2%) and bone resorption (5.35%). After multivariate analysis, intraoperative cerebrospinal fluid (CSF) leak was significantly associated with infection, whereas longer duration of surgery and unilateral site were associated with resorption. Cranioplasty using frozen autologous bone is associated with a high rate of infective complications. Intraoperative CSF leak is a potentially modifiable risk factor. Meticulous dissection during cranioplasty surgery to minimize the chance of breaching the dural or pseudodural plane may reduce the chance of bone flap. PMID:26269726

  3. Anatomic and physiological fundamentals for autologous breast reconstruction

    PubMed Central

    Mohan, Anita T.

    2015-01-01

    The success of autologous tissue transfer is reliant on adequate blood supply and as we endeavour to tailor our reconstructive options through our flap choices and design. Autologous breast reconstruction has made substantial progress over the years and the evolution of refinements over the last 30 years has allowed flaps to be based on specific perforators. The ultimate goal of breast reconstruction following mastectomy is to match optimal tissue replacement with minimal donor-site expenditure. In parallel surgeons will seek ways to ensure safe flap design and harvest while maintaining predictability and reliable tissue perfusion. Better understanding of the vascular anatomy and physiology of the cutaneous circulation of soft tissues, and that of patterns of blood flow from individual perforator has provided insight to advance perforator flap harvest and modifications in flap design. The aim of this article is to review the principles of blood supply and flap design exemplified through common flaps used in autologous breast reconstructive surgery, to better understand approaches for safe flap harvest and transfer of well perfused tissue. PMID:26005644

  4. Autologous Matrix-Induced Chondrogenesis in the Knee

    PubMed Central

    Suzer, Ferzan; Thermann, Hajo

    2014-01-01

    Objective: 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. Design and methods: 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. Results: 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. Conclusions: 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. PMID:26069694

  5. Tissue-engineered autologous grafts for facial bone reconstruction.

    PubMed

    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

    2016-06-15

    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, native bovine bone matrix, and a perfusion bioreactor for the growth and transport of living grafts, without bone morphogenetic proteins. The ramus-condyle unit, the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatán 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 nonseeded 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

  6. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity.

    PubMed

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  7. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity

    PubMed Central

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  8. The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

    PubMed

    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

    2013-04-17

    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

  9. 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

  10. Lipophosphoramidate-based bipolar amphiphiles: their syntheses and transfection properties.

    PubMed

    Berchel, Mathieu; Le Gall, Tony; Lozach, Olivier; Haelters, Jean-Pierre; Montier, Tristan; Jaffrès, Paul-Alain

    2016-03-14

    Six new cationic bolaamphiphiles (also called bipolar amphiphiles, bolaform amphiphiles, or bolalipids) were readily prepared by a thiol-ene click reaction that engaged a mercapto-alcohol (mercapto-ethanol or mercapto-hexanol) and a cationic based lipophosphoramidate. The cationic lipophosphoramidates contain two lipid chains that end in an alkene group and a selected cationic polar head group (trimethylammonium, dimethyl hydroxyethyl ammonium, or methylimidazolium). These compounds were formulated in water (with or without DOPE as a colipid) to produce supramolecular aggregates. These aggregates, before (i.e. bolasomes) and after (i.e. bolaplexes) mixing with plasmid DNA (pDNA) at various charge ratios, were characterized with regard to their sizes and zeta potentials. In the case of bolasomes, the suspensions were unstable since precipitation occurred after only a few hours at room temperature. On the other hand, bolaplex formulations exhibited clearly a better colloidal stability. Then, the gene delivery properties of the cationic bolasomes were investigated using two human-derived epithelial cell lines (A549 and 16HBE). Compared to the commercially available lipofection reagent (Lipofectamine), most of the cationic bolaamphiphiles were able to efficiently transfect these cells when they were formulated with DOPE in a 1 : 1 molar ratio. We report herein that bolaamphiphiles possessing a trimethylammonium or a dimethyl hydroxyethyl ammonium head group were the most efficient in terms of transfection efficiency while exhibiting no significant cytotoxicity. PMID:26864681

  11. In vitro transformation of mouse testis cells by oncogene transfection.

    PubMed

    Morimoto, Hiroko; Lee, Jiyoung; Tanaka, Takashi; Ishii, Kei; Toyokuni, Shinya; Kanatsu-Shinohara, Mito; Shinohara, Takashi

    2012-05-01

    Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming. PMID:22357549

  12. Altered cholesterol metabolism in APP695-transfected neuroblastoma cells.

    PubMed

    Wirths, Oliver; Thelen, Karin M; Lütjohann, Dieter; Falkai, Peter; Bayer, Thomas A

    2007-06-01

    Cholesterol has been implicated to play an important role in the generation of Abeta peptides, which are the main component of beta-amyloid plaques in the brains of patients suffering from Alzheimer's disease (AD). Epidemiological data implicate that lowering cholesterol levels has beneficial effects on the extent of beta-amyloid pathology. Thus therapeutic intervention using cholesterol lowering drugs like statins seems to be a promising approach. A couple of studies, in vitro or in vivo by the use of AD transgenic mouse models, focused on the manipulation of cholesterol levels and the resulting effects on Abeta generation. In contrast, there is not much known about the effect of the amyloid precursor protein (APP) on cholesterol levels. In the present report, we transfected human neuroblastoma cells with human APP695 and compared cellular cholesterol levels with the respective levels in Mock-transfected control cells. Furthermore, we determined the levels of diverse cholesterol precursors and metabolites using gas chromatography-mass spectrometry (GC-MS). Significant differences in the levels of the respective cholesterol precursors were observed, whereas inhibition of gamma-secretase activity by the gamma-secretase inhibitor DAPT did not have a significant effect on cellular cholesterol metabolism. PMID:17428449

  13. Autologous adipose tissue-derived mesenchymal stem cells are involved in rat liver regeneration following repeat partial hepatectomy

    PubMed Central

    LIU, TAO; MU, HONG; SHEN, ZHONGYANG; SONG, ZHUOLUN; CHEN, XIAOBO; WANG, YULIANG

    2016-01-01

    Adipose tissue-derived mesenchymal stem cells (ADSCs) have been considered to be attractive and readily available adult mesenchymal stem cells, and they are becoming increasingly popular for use in regenerative cell therapy, as they are readily accessible through minimally invasive techniques. The present study investigated whether autologous ADSC transplantation promoted liver regeneration following a repeat partial hepatectomy in rats. The rats were divided into three groups as follows: 70% partial hepatectomy (PH) group; repeat PH (R-PH) group and R-PH/ADSC group, subjected to R-PH and treated with autologous ADSCs via portal vein injection. In each group, the rats were sacrificed at different time points postoperatively in order to evaluate the changes in liver function and to estimate the liver regenerative response. The expression of proliferating cell nuclear antigen (PCNA) labeling index in the liver was measured using immunohistochemistry. The expression levels of hepatocyte growth factor (HGF) mRNA were measured using reverse transcription polymerase chain reaction. The results showed that regeneration of the remaining liver following R-PH was significantly promoted by ADSC transplantation, as shown by a significant increase in liver to body weight ratio and the PCNA labeling index at 24 h post-hepatectomy. Additionally, ADSC transplantation markedly inhibited the elevation of serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin, increased HGF content and also attenuated hepatic vacuolar degeneration 24 h postoperatively. Furthermore, the liver was found to almost fully recover from hepatocellular damage due to hepatectomy among the three groups at 168 h postoperatively. These results indicated that autologous ADSC transplantation enhanced the regenerative capacity of the remnant liver tissues in the early phase following R-PH. PMID:26783183

  14. Autologous adipose tissue‑derived mesenchymal stem cells are involved in rat liver regeneration following repeat partial hepatectomy.

    PubMed

    Liu, Tao; Mu, Hong; Shen, Zhongyang; Song, Zhuolun; Chen, Xiaobo; Wang, Yuliang

    2016-03-01

    Adipose tissue‑derived mesenchymal stem cells (ADSCs) have been considered to be attractive and readily available adult mesenchymal stem cells, and they are becoming increasingly popular for use in regenerative cell therapy, as they are readily accessible through minimally invasive techniques. The present study investigated whether autologous ADSC transplantation promoted liver regeneration following a repeat partial hepatectomy in rats. The rats were divided into three groups as follows: 70% partial hepatectomy (PH) group; repeat PH (R‑PH) group and R‑PH/ADSC group, subjected to R‑PH and treated with autologous ADSCs via portal vein injection. In each group, the rats were sacrificed at different time points postoperatively in order to evaluate the changes in liver function and to estimate the liver regenerative response. The expression of proliferating cell nuclear antigen (PCNA) labeling index in the liver was measured using immunohistochemistry. The expression levels of hepatocyte growth factor (HGF) mRNA were measured using reverse transcription polymerase chain reaction. The results showed that regeneration of the remaining liver following R‑PH was significantly promoted by ADSC transplantation, as shown by a significant increase in liver to body weight ratio and the PCNA labeling index at 24 h post‑hepatectomy. Additionally, ADSC transplantation markedly inhibited the elevation of serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin, increased HGF content and also attenuated hepatic vacuolar degeneration 24 h postoperatively. Furthermore, the liver was found to almost fully recover from hepatocellular damage due to hepatectomy among the three groups at 168 h postoperatively. These results indicated that autologous ADSC transplantation enhanced the regenerative capacity of the remnant liver tissues in the early phase following R‑PH. PMID:26783183

  15. Regional transfusion centre preoperative autologous blood donation programme: the first two years.

    PubMed Central

    Howard, M. R.; Chapman, C. E.; Dunstan, J. A.; Mitchell, C.; Lloyd, H. L.

    1992-01-01

    OBJECTIVE--To assess the efficacy of a regional autologous blood donation programme. DESIGN--Clinical and laboratory data were collected and stored prospectively. Transfusion data were collected retrospectively from hospital blood bank records. SETTING--Northern Region Blood Transfusion Service and 14 hospitals within the Northern Regional Health Authority. SUBJECTS--505 patients referred for autologous blood donation before elective surgery. MAIN OUTCOME MEASURES--Patient eligibility, adverse events from donation, autologous blood units provided, and autologous and allogeneic blood units transfused within 10 days of operation. RESULTS--Of 505 patients referred, 354 donated at least one unit. 78 of 151 referred patients who did not donate were excluded at the autologous clinic, mostly because of anaemia or ischaemic heart disease. In 73 cases the patient, general practitioner, or hospital consultant decided against donation. 363 autologous procedures were undertaken. In 213 (59%) cases all requested units were provided. The most common reasons for incomplete provision were late referral or anaemia. Adverse events accompanied 24 of 928 donations (2.6%). Transfusion data were obtained for 357 of the 363 procedures. 281 donors were transfused; autologous blood only was given to 225, autologous and allogeneic blood was given to 52, and allogeneic blood only was given to four. 648 of 902 (72%) units of autologous blood were transfused. Complete provision of requested autologous units was followed by allogeneic transfusion in 12 of 208 procedures (5.8%). Incomplete provision was followed by allogeneic transfusion in 44 of 149 procedures (30%). CONCLUSIONS--This study shows the feasibility of a regional autologous transfusion programme. Autologous donors only infrequently received allogeneic transfusion. Patients should be appropriately selected and referred early. PMID:1493393

  16. Enhanced hepatic delivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations

    PubMed Central

    Wang, Xinmei; Yu, Bo; Ren, Wei; Mo, Xiaokui; Zhou, Chenguang; He, Hongyan; Jia, HuLiang; Wang, Lu; Jacob, Samson T.; Lee, Robert J.; Ghoshal, Kalpana; Lee, L. James

    2015-01-01

    Many cationic lipids have been developed for lipid-based nanoparticles (LNPs) for delivery of siRNA and microRNA (miRNA). However, less attention has been paid to “helper lipids”. Here, we investigated several “helper lipids” and examined their effects on the physicochemical properties such as particle size and zeta potential, as well as cellular uptake and transfection efficiency. We found that inclusion of oleic acid (OA), an unsaturated fatty acid; into the LNP formulation significantly enhanced the delivery efficacy for siRNA and miRNA. For proof-of-concept, miR-122, a liver-specific microRNA associated with many liver diseases, was used as a model agent to demonstrate the hepatic delivery efficacy both in tumor cells and in animals. Compared to Lipofectamine 2000, a commercial transfection agent, OA containing LNPs delivered microRNA-122 in a more efficient manner with a 1.8-fold increase in mature miR-122 expression and a 20% decrease in Bcl-w, a target of microRNA-122. In comparison with Invivofectamine, a commercial transfection agent specifically designed for hepatic delivery, OA containing LNPs showed comparable liver accumulation and in vivo delivery efficiency. These findings demonstrated the importance of “helper lipid” components of the LNP formulation on the cellular uptake and transfection activity of siRNA and miRNA. OA containing LNPs are a promising nanocarrier system for the delivery of RNA-based therapeutics in liver diseases. PMID:24121065

  17. siRNA targeting RBP2 inhibits expression, proliferation, tumorigenicity and invasion in thyroid carcinoma cells

    PubMed Central

    KONG, LING-LING; MAN, DONG-MEI; WANG, TIAN; ZHANG, GUO-AN; CUI, WEN

    2015-01-01

    In order to estimate the effects of small interfering RNA (siRNA) targeting retinoblastoma binding protein 2 (RBP2) on the proliferation, expression, invasion, migration and tumorigenicity abilities of papillary thyroid carcinoma K1 cells, siRNA targeting RBP2 (RBP2-siRNA) and negative control siRNA were transfected into K1 cells. The mRNA levels of RBP2 in the transfected cells were estimated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein levels of RBP2 in these cells were evaluated by western blot analysis and immunocytochemical (ICC) analyses. The growth, tumorigenicity, migration and invasion abilities of the transfected cells were measured by Cell Counting Kit-8 (CCK-8), soft agar colony formation and transwell chamber assay, respectively. The ICC results demonstrated that the protein expression levels of RBP2 were lower in the RBP2-siRNA-transfected cells than in the blank and control cells (analysis of variance, F=26.754, P<0.01). RBP2-siRNA downregulated RBP2 at the mRNA (t=8.869) and protein level (F=60.835) (P=0.000 vs. control cells). In addition, the transfection of RBP2-siRNA into K1 cells also suppressed cell proliferation at 24, 48 and 72 h post-transfection (t=7.650, P<0.01; t=2.606, P=0.016; and t=2.377, P=0.027, respectively). Compared with the control group, the number of invasive and migrated cells were significantly reduced in the RBP2-siRNA-transfected group (t=4.774 and t=6.366, respectively; P<0.01). Furthermore, the tumorigenic potential of the cells transfected with RBP2-siRNA was markedly reduced, as indicated by the soft agar formation assay (t=2.749, P=0.014 vs. control cells). In conclusion, the transfection of RBP2-siRNA into papillary thyroid carcinoma K1 cells suppressed the expression of RBP2 in these cells, and reduced their proliferation, invasion, migration and tumorigenic potential. Therefore, targeting RBP2 may be an efficient approach to control thyroid carcinoma. PMID:26788140

  18. Experimental study on inhibition of the growth of human adenoid cystic cancer cells by RNA interference targeting against survivingene

    PubMed Central

    Wang, Xin; Xiong, Yu; Zhang, Congji; Zhou, Jixiang; Yang, Jun; Wang, Kun; Xia, Xiyan

    2016-01-01

    Objective: To observe the influence of RNA interference targeting against survivin gene on the biological behaviors of human adenoid cystic cancer (ACC) cells and propose the action mechanism. Method: Specific siRNA (small interfering RNA) was constructed and transfected into ACC-2 cells using liposomes. The expressions of survivin and Caspase-3 in the transfected ACC-2 cells were detected by Western Blot and RT-PCR. Cell apoptosis was detected by transmission electron microscopy, TUNEL method and flow cytometry; ultrastructural changes and cell cycles were observed. Results: Recombinant siRNA interference plasmid specifically targeting against survivin gene was constructed successfully. Survivin protein expression in the transfected ACC-2 cells was downregulated significantly, while Caspase-3 protein and mRNA expressions were upregulated and cell proliferation was inhibited considerably. Conclusion: Recombinant siRNA interference plasmid inhibited survivin mRNA and protein expressions at high efficiency, thereby inhibiting the proliferation of ACC cells. PMID:27158333

  19. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

    PubMed Central

    Pak, Jaewoo; Lee, Jung Hun; Park, Kwang Seung; Jeong, Byeong Chul; Lee, Sang Hee

    2016-01-01

    Abstract This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs) and homogenized extracellular matrix (ECM) in the form of adipose stromal vascular fraction (SVF), along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA) patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI) data, functional rating index, range of motion (ROM), and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees. PMID:27588219

  20. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix.

    PubMed

    Pak, Jaewoo; Lee, Jung Hun; Park, Kwang Seung; Jeong, Byeong Chul; Lee, Sang Hee

    2016-01-01

    This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs) and homogenized extracellular matrix (ECM) in the form of adipose stromal vascular fraction (SVF), along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA) patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI) data, functional rating index, range of motion (ROM), and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees. PMID:27588219

  1. 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.

  2. 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

  3. RNA genetics

    SciTech Connect

    Domingo, E.; Holland, J.J.; Ahlquist, P.

    1988-01-01

    These three volumes comprise reference on RNA genomes. The replication, mutation, recombination-assortment, and extreme evolutionary variability of RNA viruses and related RNA replicons is emphasized. The replication mechanisms of positive, negative, and double-stranded RNA viruses of animals and plants are featured.

  4. 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.

  5. Effect of autologous platelet-rich plasma on the chondrogenic differentiation of rabbit adipose-derived stem cells in vitro

    PubMed Central

    TANG, XIAO-BO; DONG, PEI-LONG; WANG, JIAN; ZHOU, HAI-YANG; ZHANG, HAI-XIANG; WANG, SHAN-ZHENG

    2015-01-01

    This study aimed to isolate rabbit adipose-derived stem cells (ADSCs) and explore the potential of platelet-rich plasma (PRP) in the chondrogenic differentiation of ADSCs, thereby potentially providing a new approach for the repair and regeneration of cartilage injury. Rabbit ADSCs were isolated and characterized by induction towards adipogenic, osteogenic and chondrogenic lineages in vitro. The isolated ADSCs were also cultured with or without 10% PRP. Immunofluorescence staining, toluidine blue staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect type II collagen (Col II) and aggrecan (AGC) expression. Col II immunofluorescence staining and toluidine blue staining indicated that following induction by autologous PRP, ADSCs manifested Col II and AGC expression. The expression of Col II and AGC mRNA was significantly upregulated in the PRP-treated cells when compared with that in control cells. Autologous PRP produced by laboratory centrifugation was able to promote the chondrogenic differentiation of rabbit ADSCs in vitro. PMID:26622340

  6. Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2015-01-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e. better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials. PMID:24749393

  7. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Rivoltini, L; Topalian, S L; Miki, T; Rosenberg, S A

    1994-01-01

    By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma. PMID:8170938

  8. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents

    PubMed Central

    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. PMID:26274324

  9. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  10. Ultrasound-Mediated Gene Transfection In vitro: Enhanced Efficiency by Complexation of Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Zhang, Yiwei; Tachibana, Rie; Okamoto, Akio; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Osada, Kensuke; Kataoka, Kazunori; Takagi, Shu; Matsumoto, Yoichiro

    2012-07-01

    Ultrasound-mediated gene transfection in the presence of microbubbles is a recently developed promising nonviral gene delivery method. The main obstacle towards its clinical application is its low transfection efficiency. In this work, we investigate the effect of the complexation of plasmid DNA (pDNA) into polyplex micelles on the transfection efficiency. Complexation changes the structure of pDNA and results in the condensation in size and enhanced stability. Both naked and complexed pDNAs were transfected into cultured cells using ultrasound in the presence of microbubbles. The transfection rate using complexed pDNA is considerably enhanced (from ˜0.92 to ˜1.67%, by ˜82%) compared with the rate using naked pDNA. Our method provides an alternative for the improvement of the transfection efficiency of the ultrasound-mediated method.

  11. 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.

  12. Modified gold nanoparticles for intracellular delivery of anti-liver cancer siRNA.

    PubMed

    Shaat, Hanan; Mostafa, Amany; Moustafa, Moustafa; Gamal-Eldeen, Amira; Emam, Ahmed; El-Hussieny, Enas; Elhefnawi, Mahmoud

    2016-05-17

    To overcome the rapid enzymatic degradation and low transfection efficiency of siRNA, the delivery carriers for siRNA is a therapeutic demand to increase its stability. Gold nanoparticles (AuNPs) modified by branched polyethyleneimine (bPEI) were developed as an efficient and safe intracellular delivery carriers for siRNA. The current study implied that siRNA designed against an oncogene c-Myc could be delivered by a modified AuNPs complex without significant cytotoxicity. The comparative semi-quantitative and quantitative real time PCR were used to measure the c-Myc gene expression after transfection with naked siRNA and siRNA/bPEI/AuNPs, but AuNPs interfered with PCR. However, the c-Myc protein translation was successfully detected in the transfected HuH7 cells with naked siRNA and siRNA/bPEI/AuNPs and it was found to be inhibited by siRNA/bPEI/AuNPs more than naked siRNA. The results validate the successful silencing of c-Myc gene. Accordingly, it may confirm the promising and effective delivery of siRNA by bPEI/AuNPs. The complex enhances the cellular uptake of siRNA without significant cytotoxicity and confirms that bPEI modified AuNPs could be used as a good candidate for safe cellular delivery of siRNA. PMID:27036397

  13. Highly efficient siRNA delivery system into human and murine cells using single-wall carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Ladeira, M. S.; Andrade, V. A.; Gomes, E. R. M.; Aguiar, C. J.; Moraes, E. R.; Soares, J. S.; Silva, E. E.; Lacerda, R. G.; Ladeira, L. O.; Jorio, A.; Lima, P.; Leite, M. Fatima; Resende, R. R.; Guatimosim, S.

    2010-09-01

    Development of RNA interference (RNAi) technology utilizing short interfering RNA sequences (siRNA) has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wall carbon nanotubes (SWCNTs). The CNT-siRNA delivery system successfully demonstrates nonspecific toxicity and transfection efficiency greater than 95%. This approach offers the potential for siRNA delivery into different types of cells, including hard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested the CNT-siRNA system in a non-metastatic human hepatocellular carcinoma cell line (SKHep1). In all types of cells used in this work the CNT-siRNA delivery system showed high efficiency and apparent no side effects for various in vitro applications.

  14. Targeted Surface Expression of an Exogenous Antigen in Stably Transfected Babesia bovis

    PubMed Central

    Laughery, Jacob M.; Knowles, Donald P.; Schneider, David A.; Bastos, Reginaldo G.; McElwain, Terry F.; Suarez, Carlos E.

    2014-01-01

    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. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine

  15. Targeted surface expression of an exogenous antigen in stably transfected Babesia bovis.

    PubMed

    Laughery, Jacob M; Knowles, Donald P; Schneider, David A; Bastos, Reginaldo G; McElwain, Terry F; Suarez, Carlos E

    2014-01-01

    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. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine

  16. Carotid Repair Using Autologous Adipose-Derived Endothelial Cells

    PubMed Central

    Froehlich, Harald; Gulati, Rajiv; Boilson, Barry; Witt, Tyra; Harbuzariu, Adriana; Kleppe, Laurel; Dietz, Allan B.; Lerman, Amir; Simari, Robert D.

    2009-01-01

    Background and Purpose Adipose tissue is an abundant source of endothelial cells as well as stem and progenitor cells which can develop an endothelial phenotype. It has been demonstrated that these cells have distinct angiogenic properties in vitro and in vivo. However, whether these cells have the capacity to directly improve large vessel form and function following vascular injury remains unknown. To define whether delivery of adipose-derived endothelial cells (ADECs) would improve healing of injured carotid arteries, a rabbit model of acute arterial injury was employed. Methods Autologous rabbit ADECS were generated utilizing defined culture conditions. To test the ability of ADECs to enhance carotid artery repair, cells were delivered intra-arterially following acute balloon injury. Additional delivery studies were performed following functional selection of cells prior to delivery. Results Following rabbit omental fat harvest and digestion, a proliferative, homogenous, and distinctly endothelial population of ADECs was identified. Direct delivery of autologous ADECs resulted in marked re-endothelialization 48 hours following acute vascular injury as compared to saline controls (82.2 ±26.9% vs 4.2±3.0% p<0.001). Delivery of ADECs that were selected for their ability to take up acetylated LDL significantly improved vasoreactivity and decreased intimal formation following vascular injury. Conclusions Taken together, these data suggest that ADECs represent an autologous source of proliferative endothelial cells which demonstrate the capacity to rapidly improve re-endothelialization, improve vascular reactivity, and decrease intimal formation in a carotid artery injury model. PMID:19286583

  17. Immunological aspects of allogeneic and autologous mesenchymal stem cell therapies.

    PubMed

    Hoogduijn, M J; Roemeling-van Rhijn, M; Korevaar, S S; Engela, A U; Weimar, W; Baan, C C

    2011-12-01

    Mesenchymal stem cells (MSCs) have potential for therapeutic application as an immunomodulatory and regenerative agent. The immunogenicity and survival of MSCs after infusion are, however, not clear and evidence suggests that allogeneic but also autologous MSCs disappear rapidly after infusion. This may be associated with the susceptibility of MSCs to lysis by natural killer (NK) cells, possibly a result of culture-induced stress. In the present study we examined whether NK cell-mediated lysis of MSCs could be inhibited by immunosuppressive drugs. Human MSCs were isolated from adipose tissue and expanded in culture. Peripheral blood mononuclear cells were activated with interleukin (IL)-2 (200 U/ml) and IL-15 (10 ng/ml) for 7 days. CD3(-)CD16(+)CD56(+) NK cells were then isolated by fluorescence-activated cell sorting and added to europium-labeled MSCs for 4 hr in the presence or absence of immunosuppressive drugs. Lysis of MSCs was determined by spectrophotometric measurement of europium release. Nonactivated NK cells were not capable of lysing MSCs. Cytokine-activated NK cells showed upregulated levels of granzyme B and perforin and efficiently lysed allogeneic and autologous MSCs. Addition of tacrolimus, rapamycin or sotrastaurin to the lysis assay did not inhibit MSC killing. Furthermore, preincubation of activated NK cells with the immunosuppressive drugs for 24 hr before exposure to MSCs had no effect on MSC lysis. Last, addition of the immunosuppressants before and during the activation of NK cells, reduced NK cell numbers but did not affect their capacity to lyse MSCs. We conclude that the immunosuppressive drugs tacrolimus, rapamycin, and sotrastaurin are not capable of inhibiting the lysis of allogeneic and autologous MSCs by activated NK cells. Other approaches to controlling lysis of MSCs should be investigated, as controlling lysis may determine the efficacy of MSC therapy. PMID:21732766

  18. Preclinical safety studies on autologous cultured human skin fibroblast transplantation.

    PubMed

    Zeng, Wei; Zhang, Shuying; Liu, Dai; Chai, Mi; Wang, Jiaqi; Zhao, Yuming

    2014-01-01

    Recently, FDA approved the clinical use of autologous fibroblasts (LAVIV™) for the improvement of nasolabial fold wrinkles in adults. The use of autologous fibroblasts for the augmentation of dermal and subcutaneous defects represents a potentially exciting natural alternative to the use of other filler materials for its long-term corrective ability and absence of allergic adverse effects proved by clinical application. However, compared to the clinical evidence, preclinical studies are far from enough. In this study, human skin-derived fibroblasts were cultured and expanded for both in vitro and in vivo observations. In vitro, the subcultured fibroblasts were divided into two groups. One set of cells underwent cell cycle and karyotype analysis at passages 5 and 10. The second group of cells was cocultured in medium with different concentrations of human skin extract D for the measurement of collagen concentration and cell count. In vivo, the subcultured fibroblasts were injected into nude mice subcutaneously. Biopsies were taken for morphology observation and specific collagen staining at 1, 2, and 3 months after injection. The results in vitro showed no significant differences in cell cycle distribution between passages 5 and 10. Cell proliferation and secretion were inhibited as the concentration of extract D increased. In vivo, the fibroblasts were remarkably denser on the experimental side with no dysplastic cells. Mitotic cells were easily observed at the end of the first month but were rare at the end of the third month. Type III collagen was detected at the end of the first month, while collagen type I was positive at the end of the second month. The content of both collagens increased as time passed. The above results indicated that the use of the autologous fibroblasts was safe, providing a basic support for clinical use of fibroblasts. PMID:23211390

  19. Correlating cell transfectability and motility on materials with different physico-chemical properties.

    PubMed

    Huang, Nien-Chi; Sieber, Martin; Hsu, Shan-Hui

    2015-12-01

    Gene delivery into cells can be facilitated by adding plasmid DNA/transfection reagent complexes in culture medium or pre-adsorbing the complexes on the substrate before cell seeding. Using transfection reagents, however, often causes cytotoxicity. Effective delivery of naked plasmid without any transfection reagent remains a challenge. In this study, we cultured human umbilical cord derived mesenchymal stem cells (hMSCs) on different biomaterial substrates with different physico-chemical properties and examined the transfectability of naked plasmid. Specifically, we synthesized a negatively charged polyurethane (PU) to mimic the hyaluronan-modified chitosan (CS-HA) membranes previously found to promote the transfection of naked plasmid. We observed that the PU membranes were as effective as CS-HA membranes in substrate-mediated delivery of naked plasmid into hMSCs. PU membranes with surface microgrooves further increased the gene delivery efficiency to a similar level as the commercial transfection reagent but without the harmful effect. The gene delivery efficiency was associated with the extent of activation of cellular integrins β1 and α5 on different substrates. Moreover, the delivery efficiency was positively correlated with the cell migration rate on various substrates. The substrate-mediated gene delivery by synthetic polymeric substrates supports that integrin activation and cell behavior (e.g. migration and transfectability) changes can be modulated by synthetic polymer surface with microfeatures. The transfection by PU microgrooves is easy, nontoxic, and as effective as the commercial transfection reagent. PMID:26363377

  20. Ocular toxicity following high dose chemotherapy and autologous transplant.

    PubMed

    Rubin, P; Hulette, C; Khawly, J A; Elkordy, M; Hussein, A; Vredenburgh, J J; Jaffe, G J; Peters, W P

    1996-07-01

    A 49-year-old woman received an autologous transplant for breast cancer. Six weeks later she noticed visual disturbance of the left eye which correlated with a visual field abnormality. There was a milder degree of visual disturbance in the right eye. Treatment with high-dose steroids partially stabilized the problem, which was felt to be an ischemic optic neuropathy. She ultimately died of respiratory failure. Pathology of the optic nerves revealed demyelination. Visual disturbances following high-dose chemotherapy are uncommon; the pathology to date has not been elucidated. Steroid therapy may be useful. PMID:8832031

  1. Autologous Fat Grafting: The Science Behind the Surgery.

    PubMed

    Zielins, Elizabeth R; Brett, Elizabeth A; Longaker, Michael T; Wan, Derrick C

    2016-04-01

    An invaluable part of the plastic surgeon's technical arsenal for soft tissue contouring, fat grafting continues to be plagued by unpredictable outcomes, resulting in either reoperation and/or patient dissatisfaction. Thus, extensive research has been conducted into the effects of adipose tissue procurement, processing, and placement on fat graft quality at both the cellular level and in terms of overall volume retention. Herein, we present an overview of the vast body of literature in these areas, with additional discussion of cell-assisted lipotransfer as a therapy to improve volume retention, and on the controversial use of autologous fat in the setting of prior irradiation. PMID:26961989

  2. Achieving ideal donor site aesthetics with autologous breast reconstruction

    PubMed Central

    2015-01-01

    The appearance of the donor site following breast reconstruction with abdominal flaps has become an important topic for study. Given the variety of flaps that are derived from the abdomen, decisions are often based on how much muscle and fascia will be harvested. Comparisons between muscle sparing and non-muscle sparing techniques have been performed with outcomes related to function and contour. Closure techniques will vary and include primary fascial closure, mesh reinforcement and additional fascial plication all of which can produce natural and sometimes improved abdominal contours. Proper patient selection however is important. This manuscript will describe various techniques in order to achieve ideal abdominal contour following autologous reconstruction. PMID:26005646

  3. Intramyocardial, Autologous CD34+ Cell Therapy for Refractory Angina

    PubMed Central

    Losordo, Douglas W.; Henry, Timothy D.; Davidson, Charles; Lee, Joon Sup; Costa, Marco A.; Bass, Theodore; Mendelsohn, Farrell; Fortuin, F. David; Pepine, Carl J.; Traverse, Jay H.; Amrani, David; Ewenstein, Bruce M.; Riedel, Norbert; Story, Kenneth; Barker, Kerry; Povsic, Thomas J.; Harrington, Robert A.; Schatz, Richard A.

    2011-01-01

    Rationale A growing number of patients with coronary disease have refractory angina. Preclinical and early-phase clinical data suggest that intramyocardial injection of autologous CD34+ cells can improve myocardial perfusion and function. Objective Evaluate the safety and bioactivity of intramyocardial injections of autologous CD34+ cells in patients with refractory angina who have exhausted all other treatment options. Methods and Results In this prospective, double-blind, randomized, phase II study (ClinicalTrials.gov identifier: NCT00300053), 167 patients with refractory angina received 1 of 2 doses (1×105 or 5×105 cells/kg) of mobilized autologous CD34+ cells or an equal volume of diluent (placebo). Treatment was distributed into 10 sites of ischemic, viable myocardium with a NOGA mapping injection catheter. The primary outcome measure was weekly angina frequency 6 months after treatment. Weekly angina frequency was significantly lower in the low-dose group than in placebo-treated patients at both 6 months (6.8±1.1 versus 10.9±1.2, P=0.020) and 12 months (6.3±1.2 versus 11.0±1.2, P=0.035); measurements in the high-dose group were also lower, but not significantly. Similarly, improvement in exercise tolerance was significantly greater in low-dose patients than in placebo-treated patients (6 months: 139±151 versus 69±122 seconds, P=0.014; 12 months: 140±171 versus 58±146 seconds, P=0.017) and greater, but not significantly, in the high-dose group. During cell mobilization and collection, 4.6% of patients had cardiac enzyme elevations consistent with non-ST segment elevation myocardial infarction. Mortality at 12 months was 5.4% in the placebo-treatment group with no deaths among cell-treated patients. Conclusions Patients with refractory angina who received intramyocardial injections of autologous CD34+ cells (105 cells/kg) experienced significant improvements in angina frequency and exercise tolerance. The cell-mobilization and -collection procedures

  4. Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors

    PubMed Central

    Payne, Andrew J; Gerdes, Bryan C; Kaja, Simon; Koulen, Peter

    2013-01-01

    Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during data analysis. PMID:24380024

  5. Co-delivery of DNA and siRNA via Arginine-Rich PEI-based Polyplexes

    PubMed Central

    Shan, Lu; Morris, Viola B; Labhasetwar, Vinod

    2015-01-01

    In this study, we formulated polyplexes with different compositions for co-delivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their co-delivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, co-delivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ∼100 fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects, because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression. PMID:25591125

  6. Persistent seropositivity for yellow fever in a previously vaccinated autologous hematopoietic stem cell transplantation recipient.

    PubMed

    Hayakawa, Kayoko; Takasaki, Tomohiko; Tsunemine, Hiroko; Kanagawa, Shuzo; Kutsuna, Satoshi; Takeshita, Nozomi; Mawatari, Momoko; Fujiya, Yoshihiro; Yamamoto, Kei; Ohmagari, Norio; Kato, Yasuyuki

    2015-08-01

    The duration of a protective level of yellow fever antibodies after autologous hematopoietic stem cell transplantation in a previously vaccinated person is unclear. The case of a patient who had previously been vaccinated for yellow fever and who remained seropositive for 22 months after autologous peripheral blood stem cell transplantation for malignant lymphoma is described herein. PMID:26068870

  7. Anaphylactic reaction after autologous blood transfusion: A case report and review of the literature

    PubMed Central

    Kumar, Shailendra; Goyal, Keshav; Dubey, Surya; Bindra, Ashish; Kedia, Shweta

    2015-01-01

    Autologous blood transfusion as a cause of intraoperative anaphylaxis is very rare. We encountered one such life-threatening event in a 72-year-old patient undergoing laminectomy and pedicle screw fixation. The probable cause identified was the floseal mixed autologous blood transfusion. Review of literature has been done, and measures to avoid such an event in the future are discussed. PMID:25972952

  8. Anaphylactic reaction after autologous blood transfusion: A case report and review of the literature.

    PubMed

    Kumar, Shailendra; Goyal, Keshav; Dubey, Surya; Bindra, Ashish; Kedia, Shweta

    2015-01-01

    Autologous blood transfusion as a cause of intraoperative anaphylaxis is very rare. We encountered one such life-threatening event in a 72-year-old patient undergoing laminectomy and pedicle screw fixation. The probable cause identified was the floseal mixed autologous blood transfusion. Review of literature has been done, and measures to avoid such an event in the future are discussed. PMID:25972952

  9. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    PubMed Central

    2010-01-01

    Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development. PMID:20682060

  10. Moving RNA moves RNA forward.

    PubMed

    Peng, Lina; Li, Yujiao; Zhang, Lan; Yu, Wenqiang

    2013-10-01

    Cell communication affects all aspects of cell structure and behavior, such as cell proliferation, differentiation, division, and coordination of various physiological functions. The moving RNA in plants and mammalian cells indicates that nucleic acid could be one of the various types of messengers for cell communication. The microvesicle is a critical pathway that mediates RNA moving and keeps moving RNA stable in body fluids. When moving miRNA enters the target cell, it functions by altering the gene expression profile and significantly inhibiting mRNA translation in recipient cells. Thus, moving RNA may act as a long-range modulator during development, organogenesis, and tumor metastasis. PMID:24008386

  11. Alteration of Skin Properties with Autologous Dermal Fibroblasts

    PubMed Central

    Thangapazham, Rajesh L.; Darling, Thomas N.; Meyerle, Jon

    2014-01-01

    Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices. PMID:24828202

  12. Management of Contaminated Autologous Grafts in Plastic Surgery

    PubMed Central

    Centeno, Robert F; Desai, Ankit R; Watson, Marla E

    2008-01-01

    Background: Contamination of autologous grafts unfortunately occurs in plastic surgery, but the literature provides no guidance for management of such incidents. Methods: American Society of Aesthetic Plastic Surgery members were asked to complete an online survey that asked about the number and causes of graft contaminations experienced, how surgeons dealt with the problem, the clinical outcomes, and patient disclosure. Results: Nineteen hundred surgeons were asked to participate in the survey, and 223 responded. Of these, 70% had experienced at least 1 graft contamination incident, with 26% experiencing 4 or more. The most frequently reported reason for graft contamination was a graft falling on the floor (reported by 75%). Nearly two thirds of the contaminated grafts related to craniofacial procedures. Ninety-four percent of grafts were managed with decontamination and completion of the operation. The most common method of decontamination was washing with povidone-iodine, but this practice is contrary to recommendations in the literature. Only 3 surgeons (1.9%) said a clinical infection developed following decontaminated graft use. Patients were not informed in 60% of graft contamination incidents. The survey results and review of the literature led to development of algorithms for the management of inadvertent graft contamination and patient disclosure. Conclusions: Although autologous grafts do become contaminated in plastic surgery, the overwhelming majority can be safely decontaminated and produce minimal or no clinical sequelae. The algorithms presented are intended to serve as guides for prevention of contamination events or for their management should they occur. PMID:18496583

  13. Autologous Blood Injection Works for Recalcitrant Lateral Epicondylitis

    PubMed Central

    Bostan, Bora; Balta, Orhan; Aşçı, Murat; Aytekin, Kürşad; Eser, Enes

    2016-01-01

    Background: Recalcitrant lateral epicondylitis may be a disabling condition. Treatment of this condition is still controversial. Aims: In the present prospective study, we evaluated the long-term results of autologous blood injection for the treatment of recalcitrant lateral epicondylitis. Study Design: Prospective clinical study. Methods: A total of 42 elbows of 40 consecutive patients (28 female, 12 male) were enrolled in this prospective study. Seven patients left the study (3 patients moved to another city, 1 patient died in the second week due to a heart condition, 1 patient quit the study because of the resolution of pain in the fourth week and 2 patients did not agree to the second injection). Thirteen patients were lost to third year follow-up. Therefore, a total of 21 elbows of 20 patients with 3 years of follow-up were included in this study. The mean age of the patients was 47.25 years (range, 20–68 years). Results: Visual analogue scale (VAS), Nirschl score and grip strength were significantly improved after injections when compared to before treatment. The best improvement in terms of grip strength, Nirschl score and VAS score was detected at the one year follow-up. The improvement in Nirschl and VAS score sustained until the third year. Conclusion: We suggest that autologous blood injection for the treatment of recalcitrant lateral epicondylitis is an effective, safe and successful procedure in the long-term. PMID:27403393

  14. SHIPi Enhances Autologous and Allogeneic Hematopoietic Stem Cell Transplantation

    PubMed Central

    Fernandes, Sandra; Brooks, Robert; Gumbleton, Matthew; Park, Mi-Young; Russo, Christopher M.; Howard, Kyle T.; Chisholm, John D.; Kerr, William G.

    2015-01-01

    Hematopoietic stem cell transplantation (HSCT) is a highly effective procedure enabling long-term survival for patients with hematologic malignancy or heritable defects. Although there has been a dramatic increase in the success rate of HSCT over the last two decades, HSCT can result in serious, sometimes untreatable disease due to toxic conditioning regimens and Graft-versus-Host-Disease. Studies utilizing germline knockout mice have discovered several candidate genes that could be targeted pharmacologically to create a more favorable environment for transplant success. SHIP1 deficiency permits improved engraftment of hematopoietic stem-progenitor cells (HS-PCs) and produces an immunosuppressive microenvironment ideal for incoming allogeneic grafts. The recent development of small molecule SHIP1 inhibitors has opened a different therapeutic approach by creating transient SHIP1-deficiency. Here we show that SHIP1 inhibition (SHIPi) mobilizes functional HS-PC, accelerates hematologic recovery, and enhances donor HS-PC engraftment in both allogeneic and autologous transplant settings. We also observed the expansion of key cell populations known to suppress host-reactive cells formed during engraftment. Therefore, SHIPi represents a non-toxic, new therapeutic that has significant potential to improve the success and safety of therapies that utilize autologous and allogeneic HSCT. PMID:26052545

  15. AUTOLOGOUS CHONDROCYTE TRANSPLANTATION-SERIES OF 3 CASES

    PubMed Central

    Gobbi, Riccardo Gomes; Demange, Marco Kawamura; Barreto, Ronald Bispo; Pécora, José Ricardo; Rezende, Múrcia Uchõa de; Filho, Tarcisio E.P Barros; Lombello, Christiane Bertachini

    2015-01-01

    Hyaline cartilage covers joint surfaces and plays an important role in reducing friction and mechanical loading on synovial joints such as the knee. This tissue is not supplied with blood vessels, nerves or lymphatic circulation, which may be one of the reasons why joint cartilage has such poor capacity for healing. Chondral lesions that reach the subchondral bone (osteochondral lesions) do not heal and may progress to arthrosis with the passage of time. In young patients, treatment of chondral defects of the knee is still a challenge, especially in lesions larger than 4 cm. One option for treating these patients is autologous chondrocyte transplantation/implantation. Because this treatment does not violate the subchondral bone and repairs the defect with tissue similar to hyaline cartilage, it has the theoretical advantage of being more biological, and mechanically superior, compared with other techniques. In this paper, we describe our experience with autologous chondrocyte transplantation/implantation at the Institute of Orthopedics and Traumatology, Hospital das Clínicas, University of Sâo Paulo, through a report on three cases. PMID:27022579

  16. OSTEOCHONDRAL AUTOLOGOUS TRANSPLANTATION FOR TREATING CHONDRAL LESIONS IN THE PATELLA

    PubMed Central

    Cohen, Moises; Amaro, Joicemar Tarouco; Fernandes, Ricardo de Souza Campos; Arliani, Gustavo Gonçalves; Astur, Diego da Costa; Kaleka, Camila Cohen; Skaf, Abdalla

    2015-01-01

    Objective: The primary aim of this study was to assess the clinical and functional evolution of patients with total-thickness symptomatic cartilaginous injury of the patellar joint surface, treated by means of osteochondral autologous transplantation. Methods: This prospective study was conducted from June 2008 to March 2011 and involved 17 patients. The specific questionnaires of Lysholm, Kujala and Fulkerson were completed preoperatively and one year postoperatively in order to assess the affected knee, and SF-36 was used to assess these patients’ general quality of life. The nonparametric paired Wilcoxon test was used for statistical analysis on the pre and postoperative questionnaires. The data were analyzed using the SPSS for Windows software, version 16.0, and a significance level of 5% was used. Results: The Lysholm preoperative and postoperative average scores were 54.59 and 75.76 points (p < 0.05). The Fulkerson pre and postoperative average scores were 52.53 and 78.41 points (p < 0.05). Conclusions: We believe that autologous osteochondral transplantation is a good treatment method for total-thickness symptomatic chondral lesions of the joint surface of the patella. PMID:27042645

  17. MR imaging of osteochondral grafts and autologous chondrocyte implantation

    PubMed Central

    Millington, S. A.; Szomolanyi, P.; Marlovits, S.

    2006-01-01

    Surgical articular cartilage repair therapies for cartilage defects such as osteochondral autograft transfer, autologous chondrocyte implantation (ACI) or matrix associated autologous chondrocyte transplantation (MACT) are becoming more common. MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. It should be performed with cartilage sensitive sequences, including fat-suppressed proton density-weighted T2 fast spin-echo (PD/T2-FSE) and three-dimensional gradient-echo (3D GRE) sequences, which provide good signal-to-noise and contrast-to-noise ratios. A thorough magnetic resonance (MR)-based assessment of cartilage repair tissue includes evaluations of defect filling, the surface and structure of repair tissue, the signal intensity of repair tissue and the subchondral bone status. Furthermore, in osteochondral autografts surface congruity, osseous incorporation and the donor site should be assessed. High spatial resolution is mandatory and can be achieved either by using a surface coil with a 1.5-T scanner or with a knee coil at 3 T; it is particularly important for assessing graft morphology and integration. Moreover, MR imaging facilitates assessment of complications including periosteal hypertrophy, delamination, adhesions, surface incongruence and reactive changes such as effusions and synovitis. Ongoing developments include isotropic 3D sequences, for improved morphological analysis, and in vivo biochemical imaging such as dGEMRIC, T2 mapping and diffusion-weighted imaging, which make functional analysis of cartilage possible. PMID:16802126

  18. From fresh heterologous oocyte donation to autologous oocyte banking

    PubMed Central

    Stoop, D.

    2012-01-01

    Introduction: Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. Methods: We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Results: Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women <37 years old remains constant with a mean of 4.47%. A significant proportion of young women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. Discussion and Conclusion: The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock. PMID:24753920

  19. Thermoresponsive hydrogel as a delivery scaffold for transfected rat MSCs

    PubMed Central

    Borden, Bradley A; Yockman, James; Kim, Sung Wan

    2010-01-01

    The concept of stem cells as a therapeutic agent has been gaining momentum. A common mode of administration of these cells is by direct injection into the target tissue. This can result in many of the cells being lost due to reflux from the injection site leading to a local loss of implanted cells. PoligoGel is a non-toxic hydrogel with an LCST near body temperature. It is also shown to be non-toxic to multiple cell types, and in the case of rat mesenchymal stem cells does not alter their differentiative capacity, either by inducing differentiation, or limiting the potential for subsequent differentiation after removal from the gel. Embedding cells in PoligoGel also does not interfere with the cells ability to delivery therapeutic growth factors post transfection with plasmid DNA. Here a thermoresponsive hydrogel, PoligoGel, is shown to have potential to act as a scaffold for the retention of cells at an injection site, mitigating migration or washing of the cells away from the target site after implantation. PMID:20583814

  20. 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.

  1. Dentin barrier test with transfected bovine pulp-derived cells.

    PubMed

    Schmalz, G; Schuster, U; Thonemann, B; Barth, M; Esterbauer, S

    2001-02-01

    Growth kinetics of SV40 large T-antigen-transfected bovine pulp-derived cells on dentin were investigated. These cells were used in a dentin barrier test device, and the system was evaluated by testing a set of dental filling materials. Cells (120 cells/mm2) were seeded on dentin slices and incubated for up to 21 days. Cell proliferation was recorded using MTT assay. For cytotoxicity tests 3500 cells/mm2 were seeded on dentin discs, which were then incorporated into the dentin barrier test device. After 72 h preincubation test materials were applied. After a 24 h exposure with or without perfusion of the pulpal part of the test device, cell survival was evaluated using MTT assay. The cells revealed similar growth kinetics on dentin slices and on tissue culture plates. In cytotoxicity tests the cells were more sensitive toward the test materials than previously used three-dimensional cultures of human foreskin fibroblasts and as anticipated from clinical experience. Further improvement is expected by using three-dimensional cultures of pulp-derived cells. PMID:11491647

  2. Asymmetric partitioning of transfected DNA during mammalian cell division

    PubMed Central

    Wang, Xuan; Le, Nhung; Denoth-Lippuner, Annina; Barral, Yves; Kroschewski, Ruth

    2016-01-01

    Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells. PMID:27298340

  3. Targeted surface expression of an exogenous antigen in stably transfected babesia bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

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

    PubMed

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

    2015-09-01

    The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. PMID:25645372

  5. Synergistic effect of a biosurfactant and protamine on gene transfection efficiency.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2013-04-11

    Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency. PMID:23422688

  6. Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles

    PubMed Central

    Sevcsik, Eva; Vorauer-Uhl, Karola; Lohner, Karl; Katinger, Hermann; Kunert, Renate

    2007-01-01

    At present, a number of transfection techniques are available to introduce foreign DNA into cells, but still minimal intrusion or interference with normal cell physiology, low toxicity, reproducibility, cost efficiency and successful creation of stable transfectants are highly desirable properties for improved transfection techniques. For all previous transfection experiments done in our labs, using serum-free cultivated host cell lines, an efficiency value of ∼0.1% for selection of stable cell lines has not been exceeded, consequently we developed and improved a transfection system based on defined liposomes, so-called large unilamellar vesicles, consisting of different lipid compositions to facilitate clone selection and increase the probability for creation of recombinant high-production clones. DNA and DOTAP/DOPE or CHEMS/DOPE interact by electrostatic means forming so-called lipoplexes (Even-Chen and Barenholz 2000) and the lipofection efficiency of those lipoplexes has been determined via confocal microscopy. In addition, the expression of the EGFP was determined by FACS to investigate transient as well as stable transfection and the transfection efficiency of a selection of different commercially available transfection reagents and kits has been compared to our tailor-made liposomes. PMID:19003008

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

    PubMed Central

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

    2013-01-01

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

  8. Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications.

    PubMed

    King, William J; Kouris, Nicholas A; Choi, Siyoung; Ogle, Brenda M; Murphy, William L

    2012-03-01

    Non-viral transfection is a promising technique that could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105 and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications. PMID:22277991

  9. Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications

    PubMed Central

    King, William J.; Kouris, Nicholas A.; Choi, Siyoung; Ogle, Brenda M.; Murphy, William L.

    2012-01-01

    Non-viral transfection is a promising technique which could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density, and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density, and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105, and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity, and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications. PMID:22277991

  10. Acute and persistent infection by a transfected Mo7 strain of Babesia bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stable transfection of the Mo7 strain of Babesia bovis and expression of an exogenous gene has been demonstrated in long term culture. However, the use of transfected parasites as marker vaccines or vehicles for expressing exogenous antigens in vivo requires demonstration of acute and persistent inf...

  11. Suppression of ITGB4 Gene Expression in PC-3 Cells with Short Interfering RNA Induces Changes in the Expression of β-Integrins Associated with RGD-Receptors.

    PubMed

    Knyazev, E N; Nyushko, K M; Alekseev, B Ya; Samatov, T R; Shkurnikov, M Yu

    2015-08-01

    We studied the effect of transfection of PC-3 prostate cancer cells with a plasmid encoding shRNA complimentary to a fragment of integrin β4 (ITGB4). The results attest to considerable changes in the transcriptome of transfected cells. For instance, compensatory changes in the expression of integrin family genes were found. PMID:26395630

  12. RNA epigenetics

    PubMed Central

    Liu, Nian; Pan, Tao

    2014-01-01

    Summary Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modification. PMID:24768686

  13. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  14. NBD-conjugated biosurfactant (MEL-A) shows a new pathway for transfection.

    PubMed

    Ueno, Yoshinobu; Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2007-11-20

    Gene transfection is a fundamental technology for molecular and cell biology, and also clinical gene therapy. A variety of non-viral vectors have been investigated for gene transfection, but their gene delivery had remained an inefficient process. Recently, we found that a biosurfactant, mannosylerythritol lipid (MEL)-A, dramatically increased the efficiency in transfection of plasmid DNA mediated by cationic liposomes. However, its mechanism has not been understood yet. Here we examined the mechanism of the transfection mediated by cationic liposomes with NBD-conjugated MEL-A. We found that MEL-A first gradually distributed on the intracellular membranes through the plasma membranes of target cells, while the cationic liposomes with MEL-A fused to the plasma membranes in 20-35 min. Thereafter, the oligonucleotide released from the vesicles was immediately transferred to the nucleus. The present results showed a new role of non-viral vectors in transfection. PMID:17884224

  15. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing.

    PubMed

    Woodruff, Kristina; Maerkl, Sebastian J

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  16. 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. PMID:17283962

  17. 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.

  18. Antitumoral effect of IL-12 gene transfected via liposomes into B16F0 cells.

    PubMed

    Speroni, Lucía; Gasparri, Julieta; de los A Bustuoabad, Victoria; Chiaramoni, Nadia S; Smagur, Andrzej; Szala, Stanisław; Taira, María C; del V Alonso, Silvia

    2009-01-01

    Murine melanoma B16F0 cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival. PMID:19421429

  19. Transfection of normal primary human skeletal myoblasts with p21 and p57 antisense oligonucleotides to improve their proliferation: a first step towards an alternative molecular therapy approach of Duchenne muscular dystrophy.

    PubMed

    Endesfelder, Stefanie; Bucher, Sabine; Kliche, Alexander; Reszka, Regina; Speer, Astrid

    2003-06-01

    Duchenne muscular dystrophy (DMD), caused by the absence of dystrophin, is associated with decreased muscle cell proliferation. An increased p21 mRNA level in DMD patients may be involved in the process. In this context we are interested to improve the proliferation of primary human skeletal muscle cells (SkMC) by a reduction in the cell cycle proteins p21 and p57 using the appropriate antisense oligonucleotides (ASO). Therefore a transfection procedure needs to be optimized in which the oligonucleotide enters the SkMC with a minimal loss of cell vitality and high efficiency. Three different formulations, Effectene, DAC40, and SuperFect, were compared. Proliferation was analyzed comparing cells transfected with p21 and/or p57 ASO vs. cells transfected with scrambled ASO using a bromodeoxyuridine assay. Under optimal conditions (a mixture of 0.25 microg ASO, 5 microl Effectene, 0.8 microl enhancer) SkMC transfected with p21 ASO reveal an average increase in cell proliferation of 32.5+/-11% after 24 h. p57 ASO shows the same effect, but concomitant transfection of p21 and p57 does not enhance it. A cell vitality of 78+/-14% after 24 h was determined by the MTT test. SkMC transfected with DAC40 reveal a maximal increase in proliferation of 38+/-7% after 48 h and show a vitality of 65+/-8%. In contrast to both these formulations, SuperFect was found to be highly toxic for SkMC, with more than 70% dead cells after 24 h. The increase in proliferation, the functional biological effect of p21 ASO, is well correlated with a decrease in p21 detected by western blot analysis of 31.6% for Effectene. Transfection efficiency was measured directly by FACS analysis using FITC-labeled ASO and data showing ASO internalization in 75.8+/-11.2% of the cell population for Effectene and 74.4+/-6.6% cells for DAC40. Taken together transient transfection of p21 or p57 ASO into primary human SkMC using Effectene significantly improves their proliferation compared to transfection with

  20. Enhanced transfection efficiency of poly(N,N-dimethylaminoethyl methacrylate)-based deposition transfection by combination with tris(hydroxymethyl)aminomethane.

    PubMed

    Iwai, Ryosuke; Haruki, Ryota; Nemoto, Yasushi; Nakayama, Yasuhide

    2013-02-20

    We have developed a substrate-mediated transfection method called "deposition transfection technology" using a poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics. In this study, we enhanced deposition transfection efficiency by using tris(hydroxymethyl)aminomethane (Tris buffer) as a pH adjuster for transfection solution composed of PDMAEMA and plasmid DNA (pDNA). PDMAEMA with a molecular weight of 9.7 × 10(4) g mol(-1) was synthesized by photoinduced radical polymerization. The pH of PDMAEMA solution was increased gradually in the range from 8 to 11 by the addition of Tris, and then the solubility of PDMAEMA was significantly decreased and the dissolution time was extended from 15 to 40 min at Tris/PDMAEMA ratio of 1 and higher. On the other hand, while the polyion complexes (polyplexes) were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA even under an excess amount of Tris at Tris/PDMAEMA ratio of 8, the binding affinity between PDMAEMA and pDNA was decreased with increasing Tris at Tris/PDMAEMA ratio of 2 and higher. When HeLa cells, smooth muscle cells, and cardiac fibroblasts were transfected by the deposition method using polyplex solution containing various amounts of Tris, the transgene expression dramatically increased at a Tris/PDMAEMA ratio of 2 in all cell types, which were more than 150-fold in HeLa cells, 40-fold in smooth muscle cells, and 30-fold in cardiac fibroblasts compared to those in the Tris-free condition. In addition, the enhanced transgene expression by Tris was sustained for over 10 days post-transfection as well as that observed in Tris-free condition. Thus, deposition transfection efficiency can be dramatically enhanced by using Tris buffer as a pH adjuster for polyplex solution. PMID:23360504

  1. Instability of endogenous MRP/proliferin transcripts in the nucleus of mouse embryo fibroblasts contrasts with their stability when produced during transient transfections.

    PubMed

    Malyankar, U M; Rittling, S R; Denhardt, D T

    1996-02-01

    The mitogen regulated protein/proliferin (MRP/PLF) gene is transcribed in primary mouse embryo fibroblasts (MEFs), but the pre-mRNA is not properly converted into a stable cytoplasmic mRNA and instead is rapidly degraded, apparently in the nucleus [Malyankar et al. (1994): Proc Natl Acad Sci USA 91:335-359]. In 3T3 cells derived from the MEFs by the standard 3T3 immortalization protocol, stable MRP/PLF mRNA is produced. We show here that the processing of intron sequences is similar in the two cell types and that some of the MRP/PLF transcripts are polyadenylated in the MEFs. We also document the production of stable MRP/PLF mRNA generated by transcription of various plasmid constructs containing different portions of the MRP/PLF3 gene after calcium phosphate-mediated transfection into the MEFs. We conclude that the inability of the MRP/PLF mRNA to accumulate in the MEFs is unlikely to result solely from a single localized sequence in the primary transcript (or the mRNA) that causes it to be subject to rapid breakdown; possibly export of the mRNA from the MEF nucleus is defective or some aspect of the transcriptional process marks the transcript for degradation. PMID:8655630

  2. The Efficacy and Safety of Autologous Transfusion in Unilateral Total Knee Arthroplasty

    PubMed Central

    Yoo, Moon-Jib; Ryu, Jee-Won; Kim, Jeong-Sang

    2015-01-01

    Purpose Although allogeneic blood transfusion is the most common method of transfusion in total knee arthroplasty (TKA), there are reports showing significant decrease in the amount of allogeneic transfusion and incidence of side effects after combined use of autologous transfusion. The purpose of this study is to investigate the efficacy of using an autologous transfusion device in TKA. Materials and Methods Patients who underwent TKA at our institution from January 2003 to January 2014 were divided into two groups: group A (n=127) who received allogeneic transfusion only in TKA and group B (n=118) who received autologous transfusion via an autologous transfusion device and allogeneic transfusion. In both groups, the patients were transfused when the hemoglobin level was below 9 g/dL. In group B, blood collected by the autologous transfusion device was transfused only once after surgery. The total blood loss volume, total transfusion volume, and the presence of side effects were assessed based on medical records. Results Group A received 294.6 mL more allogeneic transfusion than group B (p<0.001). There were no significant differences with regard to the development of side effects between groups. Conclusions Application of an autologous transfusion device during TKA can be effective in reducing the allogeneic transfusion volume. Moreover, allogeneic transfusion was not necessary after autologous transfusion in some patients. PMID:26389070

  3. Effect of hyperbaric oxygen therapy combined with autologous platelet concentrate applied in rabbit fibula fraction healing

    PubMed Central

    Neves, Paulo César Fagundes; de Campos Vieira Abib, Simone; Neves, Rogério Fagundes; Pircchio, Oronzo; Saad, Karen Ruggeri; Saad, Paulo Fernandes; Simões, Ricardo Santos; Moreira, Marcia Bento; de Souza Laurino, Cristiano Frota

    2013-01-01

    OBJECTIVES: The purpose is to study the effects of hyperbaric oxygen therapy and autologous platelet concentrates in healing the fibula bone of rabbits after induced fractures. METHODS: A total of 128 male New Zealand albino rabbits, between 6–8 months old, were subjected to a total osteotomy of the proximal portion of the right fibula. After surgery, the animals were divided into four groups (n = 32 each): control group, in which animals were subjected to osteotomy; autologous platelet concentrate group, in which animals were subjected to osteotomy and autologous platelet concentrate applied at the fracture site; hyperbaric oxygen group, in which animals were subjected to osteotomy and 9 consecutive daily hyperbaric oxygen therapy sessions; and autologous platelet concentrate and hyperbaric oxygen group, in which animals were subjected to osteotomy, autologous platelet concentrate applied at the fracture site, and 9 consecutive daily hyperbaric oxygen therapy sessions. Each group was divided into 4 subgroups according to a pre-determined euthanasia time points: 2, 4, 6, and 8 weeks postoperative. After euthanasia at a specific time point, the fibula containing the osseous callus was prepared histologically and stained with hematoxylin and eosin or picrosirius red. RESULTS: Autologous platelet concentrates and hyperbaric oxygen therapy, applied together or separately, increased the rate of bone healing compared with the control group. CONCLUSION: Hyperbaric oxygen therapy and autologous platelet concentrate combined increased the rate of bone healing in this experimental model. PMID:24141841

  4. Co-transplantation of autologous MSCs delays islet allograft rejection and generates a local immunoprivileged site

    PubMed Central

    Ben Nasr, Moufida; Vergani, Andrea; Avruch, James; Liu, Liye; Kefaloyianni, Eirini; D’Addio, Francesca; Tezza, Sara; Corradi, Domenico; Bassi, Roberto; Valderrama-Vasquez, Alessandro; Usuelli, Vera; Kim, James; Azzi, Jamil; Essawy, Basset El; Markmann, James; Abdi, Reza

    2016-01-01

    Aims Mesenchymal stem cells (MSCs) are multipotent cells with immunomodulatory properties. We tested the ability of MSCs to delay islet allograft rejection. Methods Mesenchymal stem cells were generated in vitro from C57BL/6 and BALB/c mice bone marrow, and their immunomodulatory properties were tested in vitro. We then tested the effect of a local or systemic administration of heterologous and autologous MSCs on graft survival in a fully allogeneic model of islet transplantation (BALB/c islets into C57BL/6 mice). Results In vitro, autologous, but not heterologous, MSCs abrogated immune cell proliferation in response to alloantigens and skewed the immune response toward a Th2 profile. A single dose of autologous MSCs co-transplanted under the kidney capsule with allogeneic islets delayed islet rejection, reduced graft infiltration, and induced long-term graft function in 30 % of recipients. Based on ex vivo analysis of recipient splenocytes, the use of autologous MSCs did not appear to have any systemic effect on the immune response toward graft alloantigens. The systemic injection of autologous MSCs or the local injection of heterologous MSCs failed to delay islet graft rejection. Conclusion Autologous, but not heterologous, MSCs showed multiple immunoregulatory properties in vitro and delayed allograft rejection in vivo when co-transplanted with islets; however, they failed to prevent rejection when injected systemically. Autologous MSCs thus appear to produce a local immunoprivileged site, which promotes graft survival. PMID:25808641

  5. Long Term Results in Refractory Tennis Elbow Using Autologous Blood

    PubMed Central

    Gani, Naseem ul; Khan, Hayat Ahmad; Kamal, Younis; Farooq, Munir; Jeelani, Hina; Shah, Adil Bashir

    2014-01-01

    Tennis elbow (TE) is one of the commonest myotendinosis. Different treatment options are available and autologous blood injection has emerged as the one of the acceptable modalities of treatment. Long term studies over a larger group of patients are however lacking. The purpose of this study was to evaluate these patients on longer durations. One-hundred and twenty patients of TE, who failed to respond to conventional treatment including local steroid injections were taken up for this prospective study over the period from year 2005 to 2011 and were followed up for the minimum of 3 years (range 3-9 years). Two mL of autologous blood was taken from the ipsilateral limb and injected into the lateral epicondyle. The effectiveness of the procedure was assessed by Pain Rating Sscale and Nirschl Staging, which was monitored before the procedure, at first week, monthly for first three months, at 6 months and then 3 monthly for first year, six monthly for next 2 years and then yearly. Statistical analysis was done and a P value of <0.05 was taken as significant. The patients (76 females and 44 males) were evaluated after procedure. The mean age group was 40.67±8.21. The mean follow up was 5.7±1.72 (range 3 to 9 years). The mean pain score and Nirschl stage before the procedure was 3.3±0.9 and 6.2±0.82 respectively. At final follow up the pain score and Nirschl were 1.1±0.9 and 1.5±0.91 respectively. Autologous blood injection was found to be one of the modalities for treatment of TE. Being cheap, available and easy method of treatment, it should be considered as a treatment modality before opting for the surgery. Universal guidelines for the management of tennis elbow should be made as there is lot of controversy regarding the treatment. PMID:25568727

  6. Long term results in refractory tennis elbow using autologous blood.

    PubMed

    Gani, Naseem Ul; Khan, Hayat Ahmad; Kamal, Younis; Farooq, Munir; Jeelani, Hina; Shah, Adil Bashir

    2014-10-27

    Tennis elbow (TE) is one of the commonest myotendinosis. Different treatment options are available and autologous blood injection has emerged as the one of the acceptable modalities of treatment. Long term studies over a larger group of patients are however lacking. The purpose of this study was to evaluate these patients on longer durations. One-hundred and twenty patients of TE, who failed to respond to conventional treatment including local steroid injections were taken up for this prospective study over the period from year 2005 to 2011 and were followed up for the minimum of 3 years (range 3-9 years). Two mL of autologous blood was taken from the ipsilateral limb and injected into the lateral epicondyle. The effectiveness of the procedure was assessed by Pain Rating Sscale and Nirschl Staging, which was monitored before the procedure, at first week, monthly for first three months, at 6 months and then 3 monthly for first year, six monthly for next 2 years and then yearly. Statistical analysis was done and a P value of <0.05 was taken as significant. The patients (76 females and 44 males) were evaluated after procedure. The mean age group was 40.67±8.21. The mean follow up was 5.7±1.72 (range 3 to 9 years). The mean pain score and Nirschl stage before the procedure was 3.3±0.9 and 6.2±0.82 respectively. At final follow up the pain score and Nirschl were 1.1±0.9 and 1.5±0.91 respectively. Autologous blood injection was found to be one of the modalities for treatment of TE. Being cheap, available and easy method of treatment, it should be considered as a treatment modality before opting for the surgery. Universal guidelines for the management of tennis elbow should be made as there is lot of controversy regarding the treatment. PMID:25568727

  7. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    PubMed

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  8. Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene

    SciTech Connect

    Nagao, Akihiro; Zhao, Xia; Takegami, Tsutomu; Nakagawa, Hideaki; Matsui, Shinobu; Matsunaga, Tsukasa; Ishigaki, Yasuhito

    2008-05-30

    To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we inserted multiple shRNA expression sequences into a single plasmid vector. In this study, the DNA repair factor XPA was selected as a target gene since it is not essential for cell viability and it is easy to check the functional knockdown of this gene. The efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of the target protein in stable transfectants, however, the multiple expression vectors apparently increased the frequency of knockdown transfectants. There were correlations between the knockdown level and marker expression in multiple-expressing transfectants, whereas poorer correlations were observed in single vector transfectants. Multiple-transfectants exhibited reduced efficiency of repair of UV-induced DNA damage and an increased sensitivity to ultraviolet light-irradiation. We propose that multiple shRNA expression vectors might be a useful strategy for establishing knockdown cells.

  9. In vivo transfection of melanoma cells by lithotripter shock waves.

    PubMed

    Bao, S; Thrall, B D; Gies, R A; Miller, D L

    1998-01-15

    The potential for gene transfection during shock wave tumor therapy was evaluated by searching for shock wave-induced DNA transfer in mouse tumor cells. B16 mouse melanoma cells were cultured by standard methods and implanted s.c. in female C57BL/6 mice 10-14 days before treatment. A luciferase reporter vector was used as the DNA plasmid for intratumoral injection at 0.2 mg/ml tumor. Air at 10% of tumor volume was injected after the DNA in some tumors to enhance acoustic cavitation activity. The shock wave generation system was similar to a Dornier HM-3 lithotripter with pressure amplitudes of 24.4 MPa peak positive and 5.2 MPa peak negative. Luciferase production in isolated tumor cells was measured with a luminometer 1 day after treatment to assess gene transfer and expression. Exposure to 800 shock waves, followed by immediate isolation and culture of tumor cells for 1 day, yielded 1.1 (0.43 SE) pg/10(6) cells for plasmid injection only and 7.5 (2.5 SE) pg/10(6) cells for plasmid plus air injection. Significantly increased luciferase production, relative to shams, occurred for 200-, 400-, 800-, and 1200-shock wave treatments with plasmid and air injection. Exposure with the isolation of tumor cells delayed for a day to allow gene expression within the growing tumors gave increased luciferase production for 100- and 400-shock wave exposures without and with air injection. Gene transfer therefore can be induced during lithotripter shock wave treatment in vivo, particularly with enhanced acoustic cavitation, which supports the concept that gene and shock wave therapy might be advantageously merged. PMID:9443395

  10. 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

  11. Effects of microRNA-21 and microRNA-24 inhibitors on neuronal apoptosis in ischemic stroke

    PubMed Central

    Liu, Wansheng; Chen, Xiaosheng; Zhang, Yu

    2016-01-01

    Objectives: The purpose of our study was aimed to investigate the effects of microRNA-21 (miR-21) and microRNA-24 (miR-24) inhibitors on ischemic stroke. Methods: MiR-21 inhibitor or miR-24 inhibitor was delivered to Sprague Dawley (SD) rats by continuous intracerebroventricular infusion. Two days later, middle cerebral artery occlusion (MCAO) was performed to induce ischemic stroke. Quantitative real-time PCR was performed to confirm transfection efficiency. The number of apoptotic neurons was detected using TUNEL method. Besides, primary hippocampal or cortical neuronal cultures were prepared from embryonic day 16-18 C57BL/6 mice. These cells were transfected with miR-21 inhibitor, miR-24 inhibitor, or negative scramble RNA. Then the cell viability was detected after transfection, as well as the protein levels of Caspase-3, B-cell lymphoma (Bcl)-xL, and heat shock protein (HSP) 70. Results: Both the levels of miR-21 and miR-24 were significantly reduced by transfection with inhibitors compared to control group or scramble RNA group (both P < 0.05). The apoptosis was significantly reduced in both hippocampal neuron and cortical neuron by miR-24 inhibitor rather than miR-21 inhibitor (P < 0.05), while the cell viability was significantly increased compared to the control group or the scramble group (P < 0.05). In addition, the levels of Bcl-xL and HSP70 were significantly increased, and the levels of Caspase-3 were statistically decreased by transfection with miR-24 inhibitor. Conclusion: MiRNA-24 but not miR-21 inhibitor prevents apoptosis in ischemic stroke by regulation of Bcl-xL, Caspase-3 and HSP70. PMID:27508039

  12. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems.

    PubMed

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  13. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

    PubMed Central

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  14. PTEN-mRNA engineered mesenchymal stem cell-mediated cytotoxic effects on U251 glioma cells

    PubMed Central

    GUO, XING RONG; HU, QIN YONG; YUAN, YA HONG; TANG, XIANG JUN; YANG, ZHUO SHUN; ZOU, DAN DAN; BIAN, LIU JIAO; DAI, LONG JUN; LI, DONG SHENG

    2016-01-01

    Mesenchymal stem cells (MSCs) have been considered to have potential as ideal carriers for the delivery of anticancer agents since the capacity for tumor-oriented migration and integration was identified. In contrast to DNA-based vectors, mRNA synthesized in vitro may be readily transfected and is mutagenesis-free. The present study was performed in order to investigate the effects of phosphatase and tensin homolog (PTEN) mRNA-engineered MSCs on human glioma U251 cells under indirect co-culture conditions. PTEN-bearing mRNA was generated by in vitro transcription and was transfected into MSCs. The expression of PTEN in transfected MSCs was detected by immunoblotting, and the migration ability of MSCs following PTEN-bearing mRNA transfection was verified using Transwell co-cultures. The indirect co-culture was used to determine the effects of PTEN-engineered MSCs on the viability of U251 glioma cells by luminescence and fluorescence microscopy. The synthesized PTEN mRNA was expressed in MSCs, and the expression was highest at 24 h subsequent to transfection. An enhanced migration rate was observed in MSCs transfected with PTEN mRNA compared with non-transfected MSCs (P<0.05). A significant inhibition of U251 cells was observed when the cells were cultured with conditioned medium from PTEN mRNA-engineered MSCs (P<0.05). The results suggested that anticancer gene-bearing mRNA synthesized in vitro is capable of being applied to a MSC-mediated anticancer strategy for the treatment of glioblastoma patients. PMID:27073544

  15. [Adenovirus-delivered BMI-1 shRNA].

    PubMed

    Chen, Zhen-Ping; Chen, Xiao-Li; Zhen, Jie

    2009-10-01

    Recently, some plasmid vectors that direct transcription of small hairpin RNAs have been developed, which are processed into functional siRNAs by cellular enzymes. Although these vectors possess certain advantages over synthesized siRNA, many disadvantages exist, including low and variable transfection efficiency. This study was aimed to establish an adenoviral siRNA delivery system without above-mentioned disadvantages on the basis of commercially available vectors. A vector was designed to target the human polycomb gene BMI-1. The pAd-BMI-1shRNA-CMV-GFP vector was produced by cloning a 300 bp U6-BMI-1 cassette from the pGE1BMI-1shRNA plasmid and a CMV-GFP cassette from pAdTrack CMV in pShutter vector. The adenovirus was produced from the 293A packaging cell line and then infected K562 cells. The mRNA and protein levels of Bmi-1 were detected by real time-PCR and Western blot respectively. The results showed that the adenovirus carrying the BMI-1shRNA was successfully produced. After being transfected with the adenovirus, the K562 cells dramatically down-regulated BMI-1 expression, whereas the adenoviruses carrying control shRNA had no effect on BMI-1 expression. It is concluded that the adenoviruses are efficient vectors for delivery of siRNA into mammalian cells and may become a candidate vector carrying siRNA drugs for gene therapy. PMID:19840467

  16. Successful autologous cord blood transplantation in a child with acquired severe aplastic anemia.

    PubMed

    Buchbinder, David; Hsieh, Loah; Puthenveetil, Geetha; Soni, Amit; Stites, Jill; Huynh, Van; Kirov, Ivan; Neudorf, Steve; Rubin, Elyssa; Sender, Leonard; Torno, Lilibeth; Margolis, David; Childs, Richard; Moore, Theodore; Nugent, Diane

    2013-05-01

    Over 400 cases of pediatric SAA occur annually in the United States. A growing number of children with SAA may have had their stem cells harvested through cord blood collection. We describe a nine-yr-old male with SAA treated successfully with an autologous cord blood transplant following immunoablative chemotherapy. With the increasing number of people cryopreserving autologous cord blood, the use of autologous cord blood in the treatment of SAA might be considered as initial therapy. This case serves to discuss approaches to preparative therapy as well as the potential complications in this growing cohort of patients. PMID:23464883

  17. Pregnancy after autologous bone marrow transplantation for malignant lymphomas.

    PubMed

    Brice, P; Pautier, P; Marolleau, J P; Castaigne, S; Gisselbrecht, C

    1994-10-01

    In the present paper, we report two cases of normal pregnancy after high dose chemotherapy and autologous stem cell transplantation (ASCT) in two of the 72 women belonging to a group of 188 patients transplanted in our unit for advanced malignant lymphoma. These pregnancies occurred 19 and 33 months after high dose therapy in two women aged 27 and 28, neither of whom had received previous total body irradiation or pelvic radiotherapy. There are several reasons for the relatively low frequency of pregnancies after ASCT. In particular, the median age of the patients is 35 years, most of these women are transplanted in relapse and have received previous pretreatment with alkylating agents and the disease free survival rate does not exceed 40%. The preservation of long term fertility should be taken into account when deciding therapeutic options for young women with curable disease. PMID:7892134

  18. Surgical management and autologous intestinal reconstruction in short bowel syndrome.

    PubMed

    Hommel, Matthijs J; van Baren, Robertine; Haveman, Jan Willem

    2016-04-01

    Short bowel syndrome (SBS) is a serious condition with considerable morbidity and mortality. When treatment with parenteral nutrition fails and life-threatening complications occur, autologous intestinal reconstruction (AIR) should be considered before intestinal transplantation (ITx). Single or combined ITx should be reserved for patients with severe liver disease and as last resort in the treatment of SBS. Longitudinal intestinal lengthening and tailoring (LILT) has proven its value in AIR, but its availability depends on the expertise of the surgeons. Serial transverse enteroplasty (STEP) has similar success rates as LILT and fewer patients progress to ITx. STEP is also applicable at small bowel dilatation in ultra-short bowel syndrome. The scope may be widened when duodenal dilatation can be treated as well. Spiral intestinal lengthening and tailoring (SILT) is a promising alternative. More research is needed to confirm these findings. Therefore we suggest an international data registry for all intestinal lengthening procedures. PMID:27086890

  19. An ethical framework for the disposal of autologous stem cells.

    PubMed

    Petrini, Carlo

    2013-01-01

    The disposal of haematopoietic stem cells stored for autologous transplantation purposes becomes a problem for hospitals when the conditions for their preservation cease to exist. When these cells have been stored for a considerable time the problem often becomes an ethical one involving informed consent and is linked to at least two simultaneous circumstances: (i) the indications regarding disposal contained in available informed consent papers are either absent or too generic; (ii) the person who provided the sample can no longer be traced. This article proposes and discusses some of the ethical criteria for addressing this problem on the basis of the so-called "principles" of North American bioethics, and compares them with some of the principles and values proposed in other models of bioethics. PMID:23412868

  20. Autologous cell therapies: challenges in US FDA regulation.

    PubMed

    McAllister, Todd N; Audley, David; L'Heureux, Nicolas

    2012-11-01

    Cell-based therapies (CBTs) have been hailed for the last two decades as the next pillar of healthcare, yet the clinical and commercial potential of regenerative medicine has yet to live up to the hype. While recent analysis has suggested that regenerative medicine is maturing into a multibillion dollar industry, examples of clinical and commercial success are still relatively rare. With 30 years of laboratory and clinical efforts fueled by countless billions in public and private funding, one must contemplate why CBTs have not made a greater impact. The current regulatory environment, with its zero-risk stance, stymies clinical innovation while fueling a potentially risky medical tourism industry. Here, we highlight the challenges the US FDA faces and present talking points for an improved regulatory framework for autologous CBTs. PMID:23210819

  1. Autologous Bone Marrow Aspirate Therapy in Wound Healing

    PubMed Central

    Chittoria, Ravi Kumar; Nandhagopal, Vijayaraghavan; Mohapatra, Devi Prasad; Thiruvoth, Friji Meethale; Sivakumar, Dinesh Kumar; Asokan, Arjun

    2016-01-01

    Objective: To study the role of autologous bone marrow aspirate therapy (ABMAT) in wound healing. Approach: This is a retrospective analysis of 9 patients (11 chronic nonhealing wounds) in whom ABMAT was used. Patients (wounds) were grouped into two groups. Group 1 included 4 patients (5 wounds) refusing/unfit for reconstruction and managed only with ABMAT. Group 2 included 5 patients (6 wounds) who agreed/fit for reconstruction after wound bed preparation with ABMAT. End point of the study was complete wound healing. Results: ABMAT helped in complete healing of chronic nonhealing wounds by secondary intention in group 1 patients and enhanced process of wound bed preparation for reconstruction in group 2 patients. Innovation: This study highlights the importance of ABMAT in the management of chronic nonhealing wounds. Conclusion: ABMAT helps in wound bed preparation to allow the wound to heal completely or cover by skin graft/flap. PMID:26989576

  2. Putting a price tag on novel autologous cellular therapies.

    PubMed

    Abou-El-Enein, Mohamed; Bauer, Gerhard; Medcalf, Nicholas; Volk, Hans-Dieter; Reinke, Petra

    2016-08-01

    Cell therapies, especially autologous therapies, pose significant challenges to researchers who wish to move from small, probably academic, methods of manufacture to full commercial scale. There is a dearth of reliable information about the costs of operation, and this makes it difficult to predict with confidence the investment needed to translate the innovations to the clinic, other than as small-scale, clinician-led prescriptions. Here, we provide an example of the results of a cost model that takes into account the fixed and variable costs of manufacture of one such therapy. We also highlight the different factors that influence the product final pricing strategy. Our findings illustrate the need for cooperative and collective action by the research community in pre-competitive research to generate the operational models that are much needed to increase confidence in process development for these advanced products. PMID:27288308

  3. Autologous Platelet Gel: Fad or Savoir? Do We Really Know?

    PubMed Central

    Stammers, Alfred H.; Trowbridge, Cody C.; Marko, Molly; Woods, Edward L.; Brindisi, Nicholas; Pezzuto, James; Klayman, Myra; Fleming, Sean; Petzold, Joseph

    2009-01-01

    Abstract: Autologous platelet-gel (APG) is the process of harvesting ones own cells (platelets), concentrating them most often through centrifugation, exposing them to an agonist which induces activation which releases intrinsic substances, and applying them to a target area to accelerate wound healing. APG is attractive because it concentrates a large number of biologically active substances, which are primarily proteins that participate in complex series of mechanisms involved in inflammation and wound healing. It has been used in numerous applications including sports medicine, dermatology, and surgery. However, there are few prospective randomized trials that have compared it in a rigorous manner to other techniques or to placebo. The following report is a review of APG, which includes a description of its perceived benefit, identification of the various modalities where it has been used, and criticisms concerning its use. PMID:20092084

  4. Stabilization of the Chest Wall: Autologous and Alloplastic Reconstructions

    PubMed Central

    Mahabir, Raman Chaos; Butler, Charles E.

    2011-01-01

    The goals of chest wall stabilization include maintenance of a rigid airtight cavity, protection of the thoracic and abdominal contents, optimization of respiration, and, whenever possible, an aesthetic reconstruction. Evidence suggests that bony fixation results in reduced ventilator dependence, a shorter overall hospital stay, and improved upper extremity function. We prefer to accomplish this with autologous tissue alone (such as the pectoralis major, latissimus dorsi, or rectus abdominus muscle flaps) for small to moderate defects. En bloc resection of defects larger than 5 cm or containing four or more ribs will likely benefit from chest wall stabilization. For patients previously treated with radiation, even larger defects may be tolerated owing to fibrosis. For these larger defects, methyl methacrylate composite meshes are used and covered with vascularized tissue. Contaminated wounds are generally reconstructed with bioprosthetic mesh rather than synthetic mesh. Using these principles, the reconstructive plastic surgeon can devise a comprehensive and safe plan to repair tremendous defects of the chest wall. PMID:22294941

  5. Ro/SSA inhibits the autologous mixed lymphocyte reaction.

    PubMed Central

    Karsh, J; Harley, J B; Goldstein, R; Lazarovits, A I

    1993-01-01

    To test the hypothesis that the Ro/SSA autoantigen can be recognized as antigenic by the human immune system, lymphocytes obtained from normal volunteers were used in in vitro assays evaluating the ability of Ro/SSA (mol. wt 60 kD) to induce B and/or T cell responses. Bovine Ro/SSA strongly inhibited the autologous mixed lymphocyte reaction in a dose-dependent manner without similar effects on concurrently performed allogeneic mixed lymphocyte reactions or T cell proliferation induced by phytohaemagglutinin. Using three colour FACS analysis, Ro/SSA was found to decrease the percentage of CD4+CD45+RA+ T cells in the proliferative, S+(G2+M), phase of the cell cycle. Associated with the decrease in the percentage of suppressor-inducer cells, was the finding that Ro/SSA was able to augment RF production in pokeweed mitogen stimulated cultures of peripheral blood lymphocytes. PMID:7678209

  6. An electroporation protocol for efficient DNA transfection in PC12 cells.

    PubMed

    Covello, Giuseppina; Siva, Kavitha; Adami, Valentina; Denti, Michela A

    2014-08-01

    A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate. PMID:23846478

  7. Serum starvation improves transient transfection efficiency in differentiating embryonic stem cells.

    PubMed

    Wallenstein, Eric J; Barminko, Jeffrey; Schloss, Rene S; Yarmush, Martin L

    2010-01-01

    Control of genetic expression is a critical issue in the field of stem cell biology, where determining a cell fate or reprogramming adult somatic cells into pluripotent cells has become a common experimental practice. In turn, for these cells to have therapeutic clinical potential, techniques for controlling gene expression are needed that minimizes or eliminates the risk of oncogenesis and mutagenesis. Possible routes for achieving this outcome could come in the form of a transient nonviral gene delivery system. In this study, we improved the efficiency of transient gene delivery to differentiating murine embryonic stem (ES) cells via serum starvation for 3 days before transfection. The transient expression of a constitutively-controlled plasmid increased from ∼50% (replated control) to ∼83% when transfected after 3 days of serum starvation but decreased to ∼28% when transfected after 3 days in normal high serum-containing media. When probed with a liver-specific reporter, Cyp7A1, expression increased from ∼1.4% (replated control) to ∼3.7% when transfected after 3 days of serum starvation but decreased to ∼0.7% when transfected after 3 days in high serum-containing media. Cy3-tagged oligonucleotides were used to rapidly quantify DNA uptake and predict ultimate transfection efficiency. This study suggests that modifications in media serum levels before transfection can have a profound effect on improving nonviral gene delivery. PMID:20574993

  8. Recording, labeling, and transfection of single neurons in deep brain structures.

    PubMed

    Dempsey, Bowen; Turner, Anita J; Le, Sheng; Sun, Qi-Jian; Bou Farah, Lama; Allen, Andrew M; Goodchild, Ann K; McMullan, Simon

    2015-01-01

    Genetic tools that permit functional or connectomic analysis of neuronal circuits are rapidly transforming neuroscience. The key to deployment of such tools is selective transfection of target neurons, but to date this has largely been achieved using transgenic animals or viral vectors that transduce subpopulations of cells chosen according to anatomical rather than functional criteria. Here, we combine single-cell transfection with conventional electrophysiological recording techniques, resulting in three novel protocols that can be used for reliable delivery of conventional dyes or genetic material in vitro and in vivo. We report that techniques based on single cell electroporation yield reproducible transfection in vitro, and offer a simple, rapid and reliable alternative to established dye-labeling techniques in vivo, but are incompatible with targeted transfection in deep brain structures. In contrast, we show that intracellular electrophoresis of plasmid DNA transfects brainstem neurons recorded up to 9 mm deep in the anesthetized rat. The protocols presented here require minimal, if any, modification to recording hardware, take seconds to deploy, and yield high recovery rates in vitro (dye labeling: 89%, plasmid transfection: 49%) and in vivo (dye labeling: 66%, plasmid transfection: 27%). They offer improved simplicity compared to the juxtacellular labeling technique and for the first time offer genetic manipulation of functionally characterized neurons in previously inaccessible brain regions. PMID:25602013

  9. Effects of molecular size and chemical factor on plasma gene transfection

    NASA Astrophysics Data System (ADS)

    Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Jinno, Masafumi

    2016-07-01

    In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.

  10. Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

    PubMed

    Jinno, Masafumi; Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu

    2016-09-01

    This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors. PMID:27136710

  11. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

    PubMed Central

    Vernon, Matthew M.; Dean, David A.; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  12. Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

    PubMed

    Chang, Lingqian; Gallego-Perez, Daniel; Zhao, Xi; Bertani, Paul; Yang, Zhaogang; Chiang, Chi-Ling; Malkoc, Veysi; Shi, Junfeng; Sen, Chandan K; Odonnell, Lynn; Yu, Jianhua; Lu, Wu; Lee, L James

    2015-08-01

    Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities. PMID:26105628

  13. Influence on the osteogenic activity of the human bone marrow mesenchymal stem cells transfected by liposome-mediated recombinant plasmid pIRES-hBMP2-hVEGF165 in vitro.

    PubMed

    Guo-ping, Wu; Xiao-chuan, He; Zhi-hui, Yang; Li, Guo

    2010-07-01

    Bone marrow mesenchymal stem cells (BMSCs) is an attractive option for use as seed cells in tissue engineering strategies. Bone morphogenetic proteins (BMPs) to be involved in the formation of various tissue types including bone, cartilage, tendon, and ligament. Vascular endothelial growth factor (VEGF) is a promising reagent for inducing angiogenesis. Constructed a coexpressing vector of human bone morphogenetic protein 2 (hBMP-2) and vascular endothelial growth factor 165 (hVEGF165). The vectors were transfected into proliferated BMSCs isolated from healthy adult bone marrow. The expression of hBMP-2 and hVEGF165 genes of BMSCs were assayed by Western-blot, and the level of alkaline phosphatase activities of BMSCs was determined by RT-PCR analysis of osteocalcin mRNA expression. The levels of collagen I by immunohistochemical staining were also determined.BMSCs transfected with reconstructed plasmid pIRES-hBMP-2-VEGF165 could secret a high level of BMP-2 and hVEGF165. The production of type I collagen, the activity of alkaline phosphatase, and the expression of osteocalcin mRNA were also significantly improved in the transfected BMSCs, compared with the control group.The exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and the lineage-committed differentiation abilities of BMSCs containing combination of genes BMP-2 and VEGF165 are enhanced. PMID:20548225

  14. Development of a Universal RNA Beacon for Exogenous Gene Detection

    PubMed Central

    Guo, Yuanjian; Lu, Zhongju; Cohen, Ira Stephen

    2015-01-01

    Stem cell therapy requires a nontoxic and high-throughput method to achieve a pure cell population to prevent teratomas that can occur if even one cell in the implant has not been transformed. A promising method to detect and separate cells expressing a particular gene is RNA beacon technology. However, developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene. PMID:25769653

  15. Synergistic effects of ultrasound-targeted microbubble destruction and TAT peptide on gene transfection: an experimental study in vitro and in vivo.

    PubMed

    Zhou, Zhiyi; Zhang, Ping; Ren, Jianli; Ran, Haitao; Zheng, Yuanyi; Li, Pan; Zhang, Qunxia; Zhang, Maohui; Wang, Zhigang

    2013-09-28

    Cell-permeable peptides (CPPs) and ultrasound-targeted microbubble destruction (UTMD) have tremendous potential for gene delivery. However, their applications are limited due to nonspecificity of CPPs and low transfection efficiency of UTMD. Here, we developed a 'smart' gene delivery system by encapsulating TAT peptide (TATp) and hepatocyte growth factor (HGF) gene within lipid microbubbles, in which TATp was protected from being enzymatically cleaved and HGF gene was protected from degradation. This new strategy had synergistic effects of UTMD and TATp on gene transfection. We investigated the efficacy and safety of HGF gene transfection mediated by the combination of UTMD and TATp in vitro and in vivo. The results from MTT assay and flow cytometry analyses indicated that the combination of UTMD and TATp could enhance HGF gene expression in HUVECs without any significant side effect on cell viability. In rat myocardial infarction models, we demonstrated that the protein and mRNA expressions of HGF in myocardium caused by the combination of UTMD and TATp were the highest. Histopathological findings demonstrated that the combination of UTMD and TATp enhanced myocardial microvasculature and ameliorated myocardial fibrosis. In conclusion, the combination of UTMD and TATp might be a safe and efficient technique for gene delivery. PMID:23791980

  16. Intracellular Delivery of siRNA by Polycationic Superparamagnetic Nanoparticles

    PubMed Central

    Castillo, Betzaida; Bromberg, Lev; López, Xaira; Badillo, Valerie; González Feliciano, Jose A.; González, Carlos I.; Hatton, T. Alan; Barletta, Gabriel

    2012-01-01

    The siRNA transfection efficiency of nanoparticles (NPs), composed of a superparamagnetic iron oxide core modified with polycationic polymers (poly(hexamethylene biguanide) or branched polyethyleneimine), were studied in CHO-K1 and HeLa cell lines. Both NPs demonstrated to be good siRNA transfection vehicles, but unmodified branched polyethyleneimine (25 kD) was superior on both cell lines. However, application of an external magnetic field during transfection (magnetofection) increased the efficiency of the superparamagnetic NPs. Furthermore, our results reveal that these NPs are less toxic towards CHO-K1 cell lines than the unmodified polycationic-branched polyethyleneimine (PEI). In general, the external magnetic field did not alter the cell's viability nor it disrupted the cell membranes, except for the poly(hexamethylene biguanide)-modified NP, where it was observed that in CHO-K1 cells application of the external magnetic field promoted membrane damage. This paper presents new polycationic superparamagnetic NPs as promising transfection vehicles for siRNA and demonstrates the advantages of magnetofection. PMID:22970377

  17. Direct Transfection of Dendritic Cells in the Epidermis After Plasmid Delivery Enhanced by Surface Electroporation

    PubMed Central

    Amante, Dinah H.; Smith, Trevor R.F.; Kiosses, Bill B.; Sardesai, Niranjan Y.; Humeau, Laurent M.P.F.

    2014-01-01

    Abstract The skin is rich in antigen-presenting cells and as such is an excellent target tissue for vaccination strategies. Electroporation is a physical delivery method that potentiates the uptake of DNA vaccines into target cells. Intradermal electroporation offers a minimally invasive solution to DNA delivery in the clinic. Here we describe the direct transfection of dendritic cells in the epidermis, using a surface dermal electroporation device, and specifically show a dendritic cell transfected with plasmid expressing green fluorescent protein. The dendritic cell has used its motile capabilities after transfection to move from the epidermis into the dermis, making its way to the lymphatic system. PMID:25470335

  18. Direct transfection of dendritic cells in the epidermis after plasmid delivery enhanced by surface electroporation.

    PubMed

    Amante, Dinah H; Smith, Trevor R F; Kiosses, Bill B; Sardesai, Niranjan Y; Humeau, Laurent M P F; Broderick, Kate E

    2014-12-01

    The skin is rich in antigen-presenting cells and as such is an excellent target tissue for vaccination strategies. Electroporation is a physical delivery method that potentiates the uptake of DNA vaccines into target cells. Intradermal electroporation offers a minimally invasive solution to DNA delivery in the clinic. Here we describe the direct transfection of dendritic cells in the epidermis, using a surface dermal electroporation device, and specifically show a dendritic cell transfected with plasmid expressing green fluorescent protein. The dendritic cell has used its motile capabilities after transfection to move from the epidermis into the dermis, making its way to the lymphatic system. PMID:25470335

  19. Assay of insulator enhancer-blocking activity with the use of transient transfection.

    PubMed

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

    2013-08-01

    We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selected from the human genome sequence. About 60-80% of the potential insulators do reveal the enhancer-blocking activity when probed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity and by dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used for quantitative analysis of the enhancer-blocking activity of the potential insulators. PMID:24228877

  20. Delivery of small interfering RNA (siRNA) using the sleeping beauty transposon.

    PubMed

    Fletcher, Bradley S

    2010-11-01

    RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. Small interfering RNAs (siRNAs) can promote sequence-specific degradation and/or translational repression of target RNA by activation of the RNA-induced silencing complex (RISC). Traditionally, silencing in mammalian cells had been achieved by transfection of synthetically derived siRNA duplexes, resulting in transient gene suppression of the target sequence. As the technology was advanced, inhibitory short-hairpin-shaped RNAs (shRNAs) could be produced by transcription from RNA polymerase-III (pol-III)-driven promoters, such as H1, U6, or cytomegalovirus (CMV)-enhanced pol III promoters. Following transcription, the shRNAs are processed by the enzyme Dicer into active siRNA. This approach allows for the continuous production of siRNA within cells using a DNA template and offers increased options for delivery of the pol-III-driven transcriptional units. A number of different viral vectors, as well as plasmid DNAs, have been utilized to deliver shRNA to mammalian cells. Here, the Tc1/mariner DNA transposon Sleeping Beauty (SB) is used as a tool to deliver shRNA-encoding transcriptional units. The SB transposon system uses a "cut-and-paste" mechanism to insert the transposon into random TA dinucleotides within the target genome. The shRNAs are then processed and used for gene knockdown. PMID:21041394

  1. Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH.

    PubMed

    Morrison, E; Tomasec, P; Linton, E A; Lowry, P J; Lowenstein, P R; Castro, M G

    1995-04-01

    Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre-proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans-Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR-CRH displayed an apparent molecular weight approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule. In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO-K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO-K1 cells is capable of stimulating ACTH release from primary cultures of anterior

  2. RNA helicases

    PubMed Central

    Ranji, Arnaz

    2010-01-01

    RNA helicases serve multiple roles at the virus-host interface. In some situations, RNA helicases are essential host factors to promote viral replication; however, in other cases they serve as a cellular sensor to trigger the antiviral state in response to viral infection. All family members share the conserved ATP-dependent catalytic core linked to different substrate recognition and protein-protein interaction domains. These flanking domains can be shuffled between different helicases to achieve functional diversity. This review summarizes recent studies, This review summarizes recent studies of RNA helicases in virus biology. First, RNA helicases are catalysts of progressive RNA-protein rearrangements that begin at gene transcription and culminate in release of infectious virus. Second, RNA helicases can act as a scaffold for alternative protein-protein interactions that can defeat the antiviral state. The mounting fundamental understanding of RNA helicases is being used to develop selective and efficacious drugs against human and animal pathogens. The analysis of RNA helicases in virus model systems continues to provide insights into virology, cell biology and immunology and has provided fresh perspective to continue unraveling the complexity of virus-host interactions. PMID:21173576

  3. RNA. Introduction.

    PubMed

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells. PMID:19263588

  4. Magna-field irradiation and autologous marrow rescue in the treatment of pediatric solid tumors

    SciTech Connect

    Munoz, L.L.; Wharam, M.D.; Kaizer, H.; Leventhal, B.G.; Ruymann, R.

    1983-12-01

    Marrow ablative therapy has been given to pediatric patients with a variety of disseminated tumors. Eight patients with advanced neuroblastoma received autologous marrow reinfusion after intensive therapy. Three of eight are in continuous complete remission from 7 to 60 months. An additional four patients received allogeneic marrow transplantation and two remain in continuous complete response at 21 and 39 months. Intensive therapy and autologous marrow reinfusion have been applied to Ewing's sarcoma, but only preliminary results are available. Six patients with disseminated rhabdomyosarcoma and extra-osseous Ewing's sarcoma received conventional chemotherapy followed by sequential hemi-body irradiation. Four of six patients received autologous marrow rescue. Their median disease-free survival is 17 months. This preliminary experience demonstrates the feasibility of using marrow ablative therapy with autologous marrow transplantation in the treatment of pediatric solid tumors. Continuing Phase II studies are required to substantiate its efficacy.

  5. Preoperative Autologous Blood Donation: Waning Indications in an Era of Improved Blood Safety.

    PubMed

    Vassallo, Ralph; Goldman, Mindy; Germain, Marc; Lozano, Miguel

    2015-10-01

    A downward trend in preoperative autologous donation (PAD) continues in Europe and the Americas, with many jurisdictions only funding medically necessary collections at present. This is the result of decreasing real and perceived residual risks of allogeneic transfusion-transmitted disease and the declining need for transfusion due to patient blood management, which have also led to escalating logistical and cost constraints for PAD programs. We outline collection trends in North America, Europe, and Latin America and review the benefits, risks, effectiveness, and safety of PAD. Important elements of informed consent follow from these points. Evidence-based medical criteria for PAD and autologous transfusion are discussed as are methods to optimize autologous collection timing to regenerate donated red cells. Recommendations for identification of patients whose risk-to-benefit ratio suggests substantial benefit compared with other autologous blood salvage and anemia management alternatives conclude the review. PMID:26006319

  6. Optimizing autologous blood donation by recombinant human erythropoietin (rhu-EPO) and interleukin 3 (IL-3).

    PubMed

    Krieter, H; Frey, L; Segiet, W; Brückner, U B; Krumwieh, D; Seiler, F R; Messmer, K

    1991-12-01

    The transfusion of autologous blood protects surgical patients from both the transfusion transmitted diseases (AIDS, posttransfusion hepatitis) and the immunosuppressive effects of homologous blood. Nevertheless, the use of autologous blood is still unsatisfactory, mainly because of the elaborated logistics, organization and technique required and the often insufficient amounts of autologous blood gained. Today, the major growth-factors of erythropoiesis are available as recombinant analogues. In the studies reviewed here, we investigated the effects of rhu-EPO and IL-3 on perioperative erythropoiesis in two canine models of acute isovolemic hemodilution. Different therapeutic concepts are compared with respect to preoperative changes in hematocrit, the volume of autologous blood gained and the duration of postdilutional anemia. PMID:1801694

  7. Comparative study of nanoparticle-mediated transfection in different GI epithelium co-culture models.

    PubMed

    Loo, Yihua; Grigsby, Christopher L; Yamanaka, Yvonne J; Chellappan, Malathi K; Jiang, Xuan; Mao, Hai-Quan; Leong, Kam W

    2012-05-30

    Oral nonviral gene delivery is the most attractive and arguably the most challenging route of administration. To identify a suitable carrier, we studied the transport of different classes (natural polymer, synthetic polymer and synthetic lipid-polymer) of DNA nanoparticles through three well-characterized cellular models of intestinal epithelium (Caco2, Caco2-HT29MTX and Caco2-Raji). Poly(phosphoramidate-dipropylamine) (PPA) and Lipid-Protamine-DNA (LPD) nanoparticles consistently showed the highest level of human insulin mRNA expression and luciferase protein expression in these models, typically at least three orders of magnitude above background. All of the nanoparticles increased tight junction permeability, with PPA and PEI having the most dramatic transepithelial electrical resistance (TEER) decreases of (35.3±8.5%) and (37.5±1.5%) respectively in the first hour. The magnitude of TEER decrease correlated with nanoparticle surface charge, implicating electrostatic interactions with the tight junction proteins. However, confocal microscopy revealed that the nanoparticles were mostly uptaken by the enterocytes. Quantitative uptake and transport experiments showed that the endocytosed, quantum dot (QD)-labeled PPA-DNA nanoparticles remained in the intestinal cells even after 24h. Negligible amount of quantum dot labeled DNA was detected in the basolateral chamber, with the exception of the Caco2-Raji co-cultures, which internalized nanoparticles 2 to 3 times more readily compared to Caco2 and Caco2-HT29MTX cultures. PEGylation decreased the transfection efficacy by at least an order of magnitude, lowered the magnitude of TEER decrease and halved the uptake of PPA-DNA nanoparticles. A key finding was insulin mRNA being detected in the underlying HepG2 cells, signifying that some of the plasmid was transported across the intestinal epithelial layer while retaining at least partial bioactivity. However, the inefficient transport suggests that transcytosis alone

  8. Biomaterials for mRNA delivery.

    PubMed

    Islam, Mohammad Ariful; Reesor, Emma K G; Xu, Yingjie; Zope, Harshal R; Zetter, Bruce R; Shi, Jinjun

    2015-12-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  9. Biomaterials for mRNA Delivery

    PubMed Central

    Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  10. Autologous versus unrelated donor allogeneic marrow transplantation for acute lymphoblastic leukemia.

    PubMed

    Weisdorf, D J; Billett, A L; Hannan, P; Ritz, J; Sallan, S E; Steinbuch, M; Ramsay, N K

    1997-10-15

    Bone marrow transplantation (BMT) can cure patients with high-risk or recurrent acute lymphoblastic leukemia (ALL). Those lacking a related donor can receive either autologous or histocompatible unrelated donor (URD) marrow. Autotransplantation may result in higher risk of relapse, whereas URD allografts, although associated with serious posttransplant toxicities, may reduce relapse risk. Six years (1987 to 1993) of consecutive autologous BMT (University of Minnesota, Dana Farber Cancer Institute; n = 214) were compared with URD transplants (National Marrow Donor Program; n = 337). Most transplants (70% autologous, 48% URD) were in early remission (first or second complete remission [CR1 or CR2]); 376 patients (75% autologous, 64% URD) were less than 18 years old. Autologous BMT led to significantly lower transplant-related mortality (TRM; relative risk [RR] 0.35; P = .001). URD transplantation offered greater protection against relapse (autologous RR 3.1; P = .001). Patients greater than 18 years old, women, and BMT recipients beyond CR2 had higher TRM, whereas adults, BMT recipients in CR2+, or BMT recipients during 1991 through 1993 had significantly more relapse. After 25 months median follow-up, 100 URD and 56 autologous recipients survive leukemia free. URD BMT in CR2 resulted in superior disease-free survival (DFS), especially for adult patients. Multivariate analysis showed superior DFS for children, men, and BMT during CR1 or 2. Autologous and URD BMT can extend survival for a minority of patients unlikely to be cured by chemotherapy, and the results with either technique are comparable. Greater toxicity and TRM after URD BMT are counterbalanced by better protection against relapse. Prospective studies addressing additional clinical variables are needed to guide clinical decision making about transplant choices for patients with ALL. PMID:9376576

  11. Multifunctional triblock copolymers for intracellular messenger RNA delivery

    PubMed Central

    Cheng, C.; Convertine, A.J.; Stayton, P.S.; Bryers, J.D.

    2012-01-01

    Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86–216 nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies. PMID:22784603

  12. Structure-activity correlation in transfection promoted by pyridinium cationic lipids.

    PubMed

    Parvizi-Bahktar, P; Mendez-Campos, J; Raju, L; Khalique, N A; Jubeli, E; Larsen, H; Nicholson, D; Pungente, M D; Fyles, T M

    2016-03-21

    The efficiency of the transfection of a plasmid DNA encoding a galactosidase promoted by a series of pyridinium lipids in mixtures with other cationic lipids and neutral lipids was assessed in CHO-K1 cells. We identify key molecular parameters of the lipids in the mixture - clog P, lipid length, partial molar volume - to predict the morphology of the lipid-DNA lipoplex and then correlate these same parameters with transfection efficiency in an in vitro assay. We define a Transfection Index that provides a linear correlation with normalized transfection efficiency over a series of 90 different lipoplex compositions. We also explore the influence of the same set of molecular parameters on the cytotoxicity of the formulations. PMID:26891970

  13. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  14. Characterization of biosurfactant-containing liposomes and their efficiency for gene transfection.

    PubMed

    Ueno, Yoshinobu; Hirashima, Naohide; Inoh, Yoshikazu; Furuno, Tadahide; Nakanishi, Mamoru

    2007-01-01

    Recently we showed significance of biosurfactants in the field of non-viral vectors for gene transfection. There, a biosurfactant, mannosylerythritol lipid A (MEL-A), especially increased the efficiency of gene transfection mediated with cationic liposomes. However, the molecular mechanism has not been well-understood yet. Here, through the examination of the ability of cationic liposomes containing an MEL (MEL-A, MEL-B or MEL-C) for important transfectional processes of the DNA capsulation and the membrane fusion with anionic liposomes, we found that MEL-A-containing liposomes increased both processes, but that MEL-B and MEL-C-containing liposomes just increased either of them. The results indicated that these kinds of the physicochemical properties in MEL-A-containing liposomes are able to increase the efficiency of liposome-mediated gene transfection. PMID:17202680

  15. DEVELOPMENT OF AN ENVIRONMENTAL ESTROGEN SCREEN USING TRANSIENTLY TRANSFECTED RAINBOW TROUT CELL LINES

    EPA Science Inventory

    Rainbow troutp hepatoma (RTH-149) and gonad cells (RTG-2) were used to develop a screening protocol for estrogen disrupting chemicals. Transfection of an estrogen-responsive luciferase reporter plasmid into...

  16. Relating Toxicity to Transfection: Using Sphingosine To Maintain Prolonged Expression in Vitro

    PubMed Central

    2015-01-01

    Cationic reagents are commonly used to facilitate DNA delivery, and transfection experiments are typically initiated in cell culture where the optimal charge ratio is determined. While transfection rates are often enhanced at higher +/– charge ratios, the cellular toxicity associated with the greater amounts of cationic components at elevated charge ratios is often not considered. In addition, the prolonged effects of cationic lipid uptake on cell viability are not evident in a typical 24–48 h transfection experiment. In this study, we compare the transfection efficiency of cationic lipoplexes to effects on viability of cultured cells in both the short and long term (7 days). Our results indicate that, while minimal toxicity is evident 24 h after exposure to DOTAP-based lipoplexes, cell viability continues to decline and ultimately compromises reporter gene expression at longer times. Substitution of a naturally occurring cationic amphiphile, sphingosine, for DOTAP greatly reduces toxicity and allows high expression to be maintained over prolonged periods. PMID:25418523

  17. Direct transfection of viral and plasmid DNA into the liver or spleen of mice.

    PubMed Central

    Dubensky, T W; Campbell, B A; Villarreal, L P

    1984-01-01

    A method for the direct transfection of polyoma viral DNA and polyoma-plasmid recombinant DNA into the liver or spleen of newborn or adult mice was developed. Calcium phosphate-precipitated DNA was injected directly into mouse organs in combination with hyaluronidase and collagenase. Transfected DNA was shown to replicate at moderate efficiency, relative to direct infection of organs with virus. Transfection with viral DNA rapidly led to an acute infection. A polyoma-bacterial plasmid recombinant DNA also was shown to replicate when transfected into mice. With this plasmid, however, genomic-length polyoma DNA rapidly recombined away from the bacterial component and replicated as viral DNA. This method should allow the direct determination of the biological activity of a cloned DNA within a mouse organ. Images PMID:6095303

  18. Usefulness of the Autologous Serum Test for the Diagnosis of Chronic Idiopathic Urticaria

    PubMed Central

    Marasoğlu Çelen, Öykü; Aydemir, Ertuğrul H.

    2014-01-01

    Background The majority of chronic urticaria cases are chronic idiopathic urticaria (CIU) with no specific identifiable etiology. The role of autoantibodies in such cases remains controversial. Objective This study determined the positivity rate of autologous serum tests in CIU patients. Methods This study was performed on 30 patients with CIU and 30 individuals without any systemic or dermatologic disease. After the volar parts of right and left forearms were cleansed, 0.05 ml serum physiologic and 0.05 ml autologous serum were injected intradermally on the right forearm 5 cm apart from each other, resulting in the formation of small papules; meanwhile, 0.05 ml histamine alone was injected to the left forearm. The test results were evaluated after 30 minutes as positive in positive cases. Results The autologous serum test produced significant and non-significant results in patients with CIU and controls, respectively. The positivity rates of the autologous serum test in the CIU and control groups were 53.3% and 26.6%, respectively. There was no relationship between autologous serum test positivity and sex in either group. In male patients with CIU, positive results ranged widely with age, while in female patients, positive results were mainly observed at younger ages with a narrow age range. Conclusion The autologous serum test is a useful test in the diagnosis and treatment of CIU as well as the selection of immunotherapy, especially in patients refractory to classic therapy. PMID:25324651

  19. Spinal fluid lymphocytes responsive to autologous and allogeneic cells in multiple sclerosis and control individuals.

    PubMed Central

    Birnbaum, G; Kotilinek, L; Schwartz, M; Sternad, M

    1984-01-01

    Spinal fluid lymphocytes from multiple sclerosis (MS) patients and controls were stimulated with either autologous non-T cells or with allogeneic non-T cells followed by stimulation with autologous non-T lymphocytes. Cells responding to these stimuli were cloned and their proliferative responses to autologous and allogeneic MS and normal non-T cells were measured. Large numbers of clones with specific patterns of reaction to both autologous and allogeneic cells were obtained from lymphocytes in MS cerebrospinal fluid (CSF), but only occasionally from cells in control CSF. Patterns of responses among clones from a particular CSF were similar and often identical, which suggested that cells in MS CSF were relatively restricted in their specificities. Surface antigen phenotyping of the clones showed them to be predominantly OKT4+, with 13% OKT8+ and 11% OKT4+8+. Peripheral T cells that were stimulated and cultured in parallel with CSF cells were different in that they usually did not give rise to as many clones nor were their patterns of response similar. Many CSF clones were heteroclitic, that is they responded to particular allogeneic cells but not autologous cells. Lymphocytes in MS CSF thus appear to represent a selected population of cells with a high frequency of responsiveness to autologous and allogeneic antigens. Such responses may be evidence for immune regulation within the central nervous system or could represent responses to altered-self antigens. PMID:6237121

  20. Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

    PubMed Central

    2002-01-01

    Background Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations. Results Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed. Conclusions Optimization of

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

    NASA Astrophysics Data System (ADS)

    Kaneko, Toshiro

    2014-10-01

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

  2. RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

    PubMed Central

    Diener, Yvonne; Jurk, Marion; Kandil, Britta; Choi, Yeong-Hoon; Wild, Stefan; Bissels, Ute; Bosio, Andreas

    2015-01-01

    Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5′- and 3′-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs. PMID:26599627

  3. Gene Transfection Method Using Atmospheric Pressure Dielectric-Barrier Discharge Plasmas

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

    2013-09-01

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

  4. Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

    PubMed

    Kusewitt, D F; Budge, C L; Nolla, H A; Edwards, B S; Ley, R D

    1992-09-01

    Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes. PMID:1380650

  5. Laser transfection with gold nanoparticles: current state and new particle structures as a perspective

    NASA Astrophysics Data System (ADS)

    Kalies, S.; Gentemann, L.; Antonopoulos, G. C.; Rakoski, M. S.; Heinemann, D.; Schomaker, M.; Ripken, T.; Meyer, H.

    2015-03-01

    Laser-based transfection techniques have gained significant interest during the last decade. Either single cell manipulation by focusing on the cell membrane or high-throughput can be realized with laser transfection. The latter is for example provided by gold nanoparticle mediated laser transfection. It is based on the heating of gold nanoparticles through laser irradiation, which permeabilizes the membrane. This technique satisfies most prerequisites of a reliable transfection technique, like efficiency and minimal cell impact. In order to bring it closer to routine usage, we investigated new particle configurations for gold nanoparticle mediated laser transfection. Our setup employs a 532 nm and 850 ps laser system. We immobilized gold particles on cell culture surfaces or modified silica particles with a gold particle surface coverage. Furthermore, first experiments achieving cell perforation with an organic nanoparticle based on polypyrrole were conducted. These three options achieved comparable efficiencies to the incubation of cells with free gold nanoparticles. With regard to the underlying mechanisms of perforation, we performed fluorescence microscopy based imaging of the cell state combined with holographic imaging directly after perforation. First results indicated a power dependent ion (calcium) and volume exchange with the extracellular medium in the first two minutes after perforation. In conclusion, our results can pave the way to a safer and more efficient way of high-throughput laser transfection with gold nanoparticles.

  6. Development of a semi-automated high throughput transient transfection system.

    PubMed

    Bos, Aaron B; Duque, Joseph N; Bhakta, Sunil; Farahi, Farzam; Chirdon, Lindsay A; Junutula, Jagath R; Harms, Peter D; Wong, Athena W

    2014-06-20

    Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody. PMID:24704608

  7. Cell Transfection with a β-Cyclodextrin-PEI-Propane-1,2,3-Triol Nanopolymer

    PubMed Central

    Lai, Wing-Fu; Jung, Han-Sung

    2014-01-01

    Successful gene therapy necessitates safe and efficient gene transfer. This article describes the use of a cationic polymer, which was synthesized by cross-linking low molecular weight branched poly(ethylenimine) (PEI) with both β-cyclodextrin and propane-1,2,3-triol, for efficient and safe non-viral gene delivery. Experimentation demonstrated that the polymer had a pH buffering capacity and DNA condensing ability comparable to those of PEI 25 kDa. In B16-F0 cells, the polymer increased the transfection efficiency of naked DNA by 700-fold and yielded better transfection efficiencies than Fugene HD (threefold higher) and PEI 25 kDa (fivefold higher). The high transfection efficiency of the polymer was not affected by the presence of serum during transfection. In addition to B16-F0 cells, the polymer enabled efficient transfection of HepG2 and U87 cells with low cytotoxicity. Our results indicated that our polymer is a safe and efficient transfection reagent that warrants further development for in vitro, in vivo and clinical applications. PMID:24956480

  8. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    NASA Astrophysics Data System (ADS)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  9. Bacterial IMPDH gene used for the selection of mammalian cell transfectants.

    SciTech Connect

    Baccam, M.; Huberman, E.; Energy Systems

    2003-06-01

    Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a hicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can he used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.

  10. Structural characterization and buffering capacity in relation to the transfection efficiency of biodegradable polyurethane.

    PubMed

    Tseng, S-ja; Tang, Shiue-cheng; Shau, Min-da; Zeng, Yi-fang; Cherng, Jong-yuh; Shih, Mei-fen

    2005-01-01

    Inefficient release of polymer/DNA complexes from endocytic vesicles into the cytoplasm and the cytotoxic nature of cationic polymers are two of the primary causes of poor gene delivery. EG-polyurethane [poly(ethylene glycol)-PU, Poly 1], EGDM-polyurethane [poly(ethylene glycol), 2-(dimethylamino)ethylamine-PU, Poly 2], and MDEADM-polyurethane [N-methyldiethanolamine, 2-(dimethylamino)ethylamine-PU, Poly 3] were designed in this study to overcome these obstacles. The structural characteristics of polyurethanes and physicochemical properties of their formed complexes with DNA were determined to correlate their transfection efficiency. The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than those of Poly 2 and Poly 1. This study reveals that the amine groups in the polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Also the size and binding properties of DNA and polymeric transfectants can be in correlation to the transfection efficiency of resulting DNA/polymer complexes. PMID:16287233

  11. Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids.

    PubMed

    Tucker, Torry A; Varga, Karoly; Bebok, Zsuzsa; Zsembery, Akos; McCarty, Nael A; Collawn, James F; Schwiebert, Erik M; Schwiebert, Lisa M

    2003-03-01

    Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function. PMID:12421695

  12. Influence of the H2O2 in the plasma gene transfection method

    NASA Astrophysics Data System (ADS)

    Kimura, Masanori; Tachibana, Hiroki; Ohno, Yuki; Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Tachibana, Kunihide; Jinno, Masafumi

    2015-09-01

    Gene transfection is the process of deliberately introducing nucleic acids into cells. The authors have been developing a novel gene transfection method using microplasma irradiation (plasma gene transfection method). Our previous study shows that long life chemically reactive species contribute to gene transfection, which induce the transfection at least 60 s after plasma irradiation (after effect). In order to clarify the key reactive species which is effective on the after effect, the effect of H2O2 addition after plasma irradiation was investigated. Addition of H2O2 at 1/1000 -1 ppm after plasma irradiation did not largely affect or slightly decease the transfection ratio, whereas the H2O2 concentration induced by plasma irradiation is estimated as 2.7 ppb after dilution by the medium. It is found that the H2O2 is not main species for the after effect. This work was partly supported by JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (Number 25108509, 15H00896) and a grant from Ehime University.

  13. A Method to Evaluate the Efficiency of Transfection Reagents in an Adherent Zebrafish Cell Line

    PubMed Central

    Aschberger, Teresa; Pelster, Bernd

    2013-01-01

    Abstract We present a simple and robust method to evaluate the transfection efficiency of commercially available transfection reagents intended to be established for use in nonmammalian cell lines. To illustrate the method, we compare the ability of four different reagents to transfect the embryonic zebrafish cell line Z3. Z3 cells were seeded in a 96-well plate and simultaneously transfected in several variations by using minimum volumes of transfection reagent and a vector DNA encoding an amplified version of green fluorescent protein (GFP). After 24 and 48 h, transfection efficiency was determined by a dual fluorescence plate reader measurement of GFP and Hoechst 33342 fluorescence, an indicator of cell density. Of the four different reagents tested, certain variations of JetPrime™ reagent and X-tremeGene™ HP reagent produced the highest fluorescence signal per cell after 24- and 48-h incubation, respectively. The simultaneous multivariate setup enables comparing different reagent/DNA combinations at different time points well, independent of cell growth variability or seeding density. PMID:23515475

  14. Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles

    PubMed Central

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

    2013-01-01

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

  15. Efficient telomerase inhibition in human non-small cell lung cancer cells by liposomal delivery of 2'-O-methyl-RNA.

    PubMed

    Beisner, Julia; Dong, Meng; Taetz, Sebastian; Piotrowska, Kamilla; Kleideiter, Elke; Friedel, Godehard; Schaefer, Ulrich; Lehr, Claus-Michael; Klotz, Ulrich; Mürdter, Thomas E

    2009-05-01

    The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase. PMID:18803262

  16. Use of autologous growth factors in lumbar spinal fusion.

    PubMed

    Lowery, G L; Kulkarni, S; Pennisi, A E

    1999-08-01

    The results of spinal fusion, especially posteriorly above the lumbosacral junction, have been mixed. Autologous growth factor concentrate (AGF) prepared by ultraconcentration of platelets contains multiple growth factors having a chemotactic and mitogenic effect on mesenchymal stem cells and osteoblasts and may play a role in initiating bone healing. The purpose of this retrospective study is to review our results with AGF in lumbar spinal fusions. To date, AGF has been used in 39 patients having lumbar spinal fusion. The study group consisted of the first 19 consecutive cases to allow at least 6 months follow-up. The average follow-up was 13 months (range 6 to 18 months). Follow-up compliance was 91%. There were 7 men and 12 women. Average age was 52 years (range 30-72 years). Nine patients had prior back surgery. There were 8 smokers. AGF was used in posterior (n = 15) or anterior intradiscal (n = 4) fusions. AGF was used with autograft and coraline hydroxyapatite in all posterior fusions, and autograft, coral, and intradiscal spacer (carbon fiber spinal fusion cages or Synthes femoral ring) in intradiscal fusions. Posterior stabilization was used in all cases. Eight cases were single-level fusions, 6 were two-level, and 1 was a three-level fusion. Autologous iliac crest bone graft was taken in 14 cases and local autograft used in 5 cases. Posteriorly, a total of 23 levels were fused; of these, nine were at L5-S1, eight at L4-L5, five at L3-L4, and one at L2-L3. No impending pseudoarthroses were noted on plain radiographic examination at last follow-up visit. Solid fusion was confirmed in 3 patients having routine hardware removal, and in 2 patients who had surgery at an adjacent level. There was one posterior wound infection, which was managed without sequelae. When used as an adjunct to autograft, AGF offers theoretical advantages that need to be examined in controlled studies. Further study is necessary to determine whether coralline hydroxyapatite used as a

  17. Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

    PubMed

    Yu, G; Okawa, H; Okita, K; Kamano, Y; Wang, F; Saeki, M; Yatani, H; Egusa, H

    2016-01-01

    Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of

  18. Cell-based assay system for high-throughput screening of anti-photo-aging agents in fibroblast transfectants.

    PubMed

    Lee, S; Shin, S; Jung, E; Park, D

    2016-08-01

    The matricellular protein CCN1 is significantly elevated in acutely ultraviolet-irradiated human skin and negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation. In this study, we established a stable cell line, termed CCN1-GFs, by transfection of the pAcGFP1-1-CCN1 promoter plasmid and examined its usefulness as a cell-based assay system for screening anti-aging ingredients. The promoter of the reporter plasmid pAcGFP1-1-CCN1 promoter was transfected into NIH3T3 cells using the Lipofectamine reagent. G418-resistant cells were selected and further cloned. To confirm whether AcGFP1-1-CCN1 promoter plasmid recombined in the NIH3T3 cells, the level of AcGFP1-1-CCN1 was measured by PCR analysis. To determine if NIH3T3 cells expressed the gene encoding green fluorescence protein in a CCN1 promoter-dependent manner, the reporter enzyme activities were assayed using a fluorimeter and flow cytometer. To determine if CCN1 inhibitor, which was selected through this system, exerted a direct effect on the downstream signal, mRNA expression of collagen1 and MMP1A was checked by using real-time PCR. UVB irradiation of CCN1-GFs resulted in increased CCN1 promoter activity. Treatment with retinoic acid, a CCN1 inhibitor, inhibited UV-induced CCN1 promoter activity. Subsequent use of this assay system to screen anti-aging ingredients revealed that CCN1-GFs treated with sclareol showed decreased levels of UVB-induced CCN1 expression. Sclareol attenuated UVB-induced photo-aging by an increase in collagen synthesis and decrease in MMP-1 activity. PMID:26281901

  19. Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.

    PubMed

    Bradley, Todd; Fera, Daniela; Bhiman, Jinal; Eslamizar, Leila; Lu, Xiaozhi; Anasti, Kara; Zhang, Ruijung; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Stolarchuk, Christina; Lloyd, Krissey E; Parks, Robert; Eaton, Amanda; Foulger, Andrew; Nie, Xiaoyan; Karim, Salim S Abdool; Barnett, Susan; Kelsoe, Garnett; Kepler, Thomas B; Alam, S Munir; Montefiori, David C; Moody, M Anthony; Liao, Hua-Xin; Morris, Lynn; Santra, Sampa; Harrison, Stephen C; Haynes, Barton F

    2016-01-01

    Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization. PMID:26725118

  20. Identification of factors involved in target RNA-directed microRNA degradation.

    PubMed

    Haas, Gabrielle; Cetin, Semih; Messmer, Mélanie; Chane-Woon-Ming, Béatrice; Terenzi, Olivier; Chicher, Johana; Kuhn, Lauriane; Hammann, Philippe; Pfeffer, Sébastien

    2016-04-01

    The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3'-5' exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection. PMID:26809675

  1. A capsidless ssRNA virus hosted by an unrelated dsRNA virus.

    PubMed

    Zhang, Rui; Hisano, Sakae; Tani, Akio; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2016-01-01

    Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus. PMID:27571749

  2. Identification of factors involved in target RNA-directed microRNA degradation

    PubMed Central

    Haas, Gabrielle; Cetin, Semih; Messmer, Mélanie; Chane-Woon-Ming, Béatrice; Terenzi, Olivier; Chicher, Johana; Kuhn, Lauriane; Hammann, Philippe; Pfeffer, Sébastien

    2016-01-01

    The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection. PMID:26809675

  3. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    SciTech Connect

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-07-24

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43/sup 0/C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37/sup 0/C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 ..mu..g per 10/sup 9/ cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs.

  4. RNA Research

    NASA Technical Reports Server (NTRS)

    1998-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. It is widely believed that this RNA World was extensive and therefore a sophisticated nucleic acid replication machinery would presumably predate the translation machinery which would not be needed until later stages in the development of life. This view of an extended RNA World is not necessarily correct. From the point of view of exobiology, the difference in these two views mainly affects the significance of studies of the extent of catalysis possible by RNA- In either case, the origin of the translation machinery and the principles of RNA evolution remain central problems in exobiology. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modem organisms came to exist by the time of the last common ancestor (as detected by 16S RRNA sequence studies). Third, the RNAs that comprise the ribosome are themselves likely of very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.

  5. Rapid, in vivo, evaluation of antiangiogenic and antineoplastic gene products by nonviral transfection of tumor cells.

    PubMed

    Weiss, Jonathan M; Shivakumar, Rama; Feller, Stephanie; Li, Lin-Hong; Hanson, Art; Fogler, William E; Fratantoni, Joseph C; Liu, Linda N

    2004-05-01

    Using a nonviral, electroporation-based gene transfection approach, we demonstrate the efficient and consistent transfection of two poorly immunogenic tumor cell lines: B16F10 melanoma and renal carcinoma (RENCA). Three genes, IL-12, angiostatin (AS), and an endostatin:angiostatin fusion protein (ES:AS) were subcloned into a DNA plasmid containing EBNA1-OriP, which was then transfected into B16F10 and RENCA cells. Significant levels of protein were secreted into the culture supernatants of transfected cells in vitro. Transfected tumor cells were injected subcutaneously into mice. All the three transgenes were capable of significantly delaying and reducing the formation of primary B16F10 and RENCA tumors, as well as B16F10 lung metastases. By day 11 post-injection, all control mice that received either mock-transfected or empty vector DNA-transfected B16F10 tumor cells had developed large primary tumors. In contrast, mice that received IL-12-transfected B16F10 cells did not develop appreciable tumors until day 17, and these were significantly smaller than controls. Similar results were observed for the RENCA model, in which only one of the IL-12 mice had developed tumors out to day 31. Expression of AS or ES:AS also significantly delayed and reduced primary tumors. Overall, ES:AS was more effective than AS alone. Furthermore, 25% of the AS mice and 33% of the ES:AS mice remained tumor-free at day 17, by which point all control mice had significant tumors. Mouse survival rates also correlated with the extent of tumor burden. Importantly, no lung metastases were detected in the lungs of mice that had received either AS or ES:AS-transfected B16F10 tumor cells and significantly fewer metastases were found in the IL-12 group. The consistency of our transfection results highlight the feasibility of directly electroporating tumor cells as a means to screen, identify, and validate in vivo potentially novel antiangiogenic and/or antineoplastic genes. PMID:15031722

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

  7. RNA helicase A is not required for RISC activity.

    PubMed

    Liang, Xue-Hai; Crooke, Stanley T

    2013-10-01

    It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects. PMID:23895878

  8. Endocrinopathies after allogeneic and autologous transplantation of hematopoietic stem cells.

    PubMed

    Orio, Francesco; Muscogiuri, Giovanna; Palomba, Stefano; Serio, Bianca; Sessa, Mariarosaria; Giudice, Valentina; Ferrara, Idalucia; Tauchmanovà, Libuse; Colao, Annamaria; Selleri, Carmine

    2014-01-01

    Early and late endocrine disorders are among the most common complications in survivors after hematopoietic allogeneic- (allo-) and autologous- (auto-) stem cell transplant (HSCT). This review summarizes main endocrine disorders reported in literature and observed in our center as consequence of auto- and allo-HSCT and outlines current options for their management. Gonadal impairment has been found early in approximately two-thirds of auto- and allo-HSCT patients: 90-99% of women and 60-90% of men. Dysfunctions of the hypothalamus-pituitary-growth hormone/insulin growth factor-I axis, hypothalamus-pituitary-thyroid axis, and hypothalamus-pituitary-adrenal axis were documented as later complicances, occurring in about 10, 30, and 40-50% of transplanted patients, respectively. Moreover, overt or subclinical thyroid complications (including persistent low-T3 syndrome, chronic thyroiditis, subclinical hypo- or hyperthyroidism, and thyroid carcinoma), gonadal failure, and adrenal insufficiency may persist many years after HSCT. Our analysis further provides evidence that main recognized risk factors for endocrine complications after HSCT are the underlying disease, previous pretransplant therapies, the age at HSCT, gender, total body irradiation, posttransplant derangement of immune system, and in the allogeneic setting, the presence of graft-versus-host disease requiring prolonged steroid treatment. Early identification of endocrine complications can greatly improve the quality of life of long-term survivors after HSCT. PMID:24883377

  9. Pediatric penile reconstruction using autologous split-thickness skin graft.

    PubMed

    Diaz, E C; Corcoran, J F; Johnson, E K

    2016-06-01

    This video provides a case report of penis entrapment secondary to excessive skin removal during circumcision. It highlights the technical aspects of pediatric penile reconstruction using autologous split-thickness skin graft (STSG). Key points include: 1. Infection prevention is paramount and antibiotic prophylaxis is routine. 2. The usual harvest site for the STSG is the lateral thigh because of its source of glabrous skin and convenient proximity to the penis. The lateral thigh is also outside of the diapered area, which helps lessen postoperative pain and infectious risks. 3. A dermatome is used to harvest the STSG. Skin thickness for penis coverage at this age is usually 10-12/1000 of an inch. 4. Direct contact of the graft and wound bed is essential for graft uptake. Hemostasis of the wound bed is critical to prevent hematoma formation. Elimination of redundant tissue is also important to ensure maximal contact between the graft and underlying wound bed. 5. A pressure dressing or bolster is used to prevent shear, and provide contact between the graft and wound bed for at least the first 5 days. 6. A semi-occlusive dressing, Tegaderm, was used on the donor site and it is believed that it provides a moist environment conducive for epithelial and dermal healing. 7. Lymphedema can result if excess distal penile skin is not excised. It is prudent to limit the amount of mucosal collar or consider direct anastomosis to the glans. PMID:27155806

  10. COMPARISON OF TWIN AND AUTOLOGOUS TRANSPLANTS FOR MULTIPLE MYELOMA

    PubMed Central

    Bashey, Asad; Pérez, Waleska S.; Zhang, Mei-Jie; Anderson, Kenneth C.; Ballen, Karen; Berenson, James R.; To, L. Bik; Fonseca, Rafael; Freytes, César O.; Gale, Robert Peter; Gibson, John; Giralt, Sergio A.; Kyle, Robert A.; Lazarus, Hillard M.; Maharaj, Dipnarine; McCarthy, Philip L.; Milone, Gustavo A.; Nimer, Stephen; Pavlovsky, Santiago; Reece, Donna E.; Schiller, Gary; Vesole, David H.; Hari, Parameswaran

    2008-01-01

    Relapse is the overwhelming cause of treatment-failure after autologous transplantation for multiple myeloma (MM). For patients with a syngeneic donor, twin transplants provide a healthy graft that is free of myeloma. The relative impact of the graft on post-transplant relapse can be estimated by comparing risk of relapse after hematopoietic cell transplantation from genetically-identical twins vs. autotransplants since confounding differences in minor or major histocompatibility antigens are absent in the syngeneic transplant setting. Outcomes of 43 subjects who received twin transplants for MM were compared to 170 matched autotransplant recipients reported to the CIBMTR. Multivariate analysis was performed by fitting a Cox model stratified on matched-pairs. The matched transplant patients studied were similar with respect to subject-, disease- and transplant-related characteristics. Cumulative incidence of relapse/progression was significantly lower and progression-free survival was significantly higher following twin transplants. In multivariate analysis, the probability of relapse/progression was lower in twins (relative risk, RR=0.49, 95% confidence interval (CI) 0.28 – 0.86, p=0.011). Twin transplants have a significantly lower relapse risk than autotransplants in multiple myeloma suggesting that graft composition may impact outcomes following high-dose chemotherapy. PMID:18804041

  11. Hepcidin as a new biomarker for detecting autologous blood transfusion.

    PubMed

    Leuenberger, Nicolas; Barras, Laura; Nicoli, Raul; Robinson, Neil; Baume, Norbert; Lion, Niels; Barelli, Stefano; Tissot, Jean-Daniel; Saugy, Martial

    2016-05-01

    Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hr and 1 day after blood reinfusion, these concentrations rose by seven- and fourfold, respectively. No significant change was observed in the control phase. Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT. Am. J. Hematol. 91:467-472, 2016. © 2016 Wiley Periodicals, Inc. PMID:26822428

  12. Autologous transplant in multiple myeloma with an augmented conditioning protocol.

    PubMed

    Abu Zaid, Badran; Abdul-Hai, Ali; Grotto, Itamar; Dray, Lillian; Resnick, Igor B; Tsirigotis, Panagiotis D; Samuel, Simcha; Or, Reuven; Shapira, Michael Y

    2013-11-01

    We compared the tolerability and anti-myeloma effect of two conditioning regimens for autologous stem cell transplant (auto-SCT) in consecutive groups of patients. Protocol 1 was the earlier, and consisted of the combination of three agents in a sequential manner, including etoposide, thiotepa and melphalan (n = 29), while protocol 2 employed melphalan alone (n = 34). The two groups were comparable (other than younger age in protocol 1). Conditioning with protocol 1 seemed more toxic, as expressed by the higher number of febrile days and higher demand for parenteral nutrition. This was not expressed with longer admission time. With 108 and 60 months' median follow-up, respectively, the median survival in patients treated by protocol 2 (melphalan 200 mg/m(2)) was reached at 59 months, while the median survival was not yet reached in patients treated with protocol 1 (p = 0.039). The time to progression was significantly longer with protocol 1 (median 44 months vs. 17 months with protocol 2, p = 0.033). Confounded by the small number of patients, conditioning with melphalan augmented by etoposide and thiotepa in a sequential manner is slightly more toxic than melphalan alone and may benefit patients with myeloma undergoing auto-SCT. PMID:23469964

  13. Endocrinopathies after Allogeneic and Autologous Transplantation of Hematopoietic Stem Cells

    PubMed Central

    Muscogiuri, Giovanna; Palomba, Stefano; Serio, Bianca; Sessa, Mariarosaria; Giudice, Valentina; Ferrara, Idalucia; Tauchmanovà, Libuse; Colao, Annamaria; Selleri, Carmine

    2014-01-01

    Early and late endocrine disorders are among the most common complications in survivors after hematopoietic allogeneic- (allo-) and autologous- (auto-) stem cell transplant (HSCT). This review summarizes main endocrine disorders reported in literature and observed in our center as consequence of auto- and allo-HSCT and outlines current options for their management. Gonadal impairment has been found early in approximately two-thirds of auto- and allo-HSCT patients: 90–99% of women and 60–90% of men. Dysfunctions of the hypothalamus-pituitary-growth hormone/insulin growth factor-I axis, hypothalamus-pituitary-thyroid axis, and hypothalamus-pituitary-adrenal axis were documented as later complicances, occurring in about 10, 30, and 40–50% of transplanted patients, respectively. Moreover, overt or subclinical thyroid complications (including persistent low-T3 syndrome, chronic thyroiditis, subclinical hypo- or hyperthyroidism, and thyroid carcinoma), gonadal failure, and adrenal insufficiency may persist many years after HSCT. Our analysis further provides evidence that main recognized risk factors for endocrine complications after HSCT are the underlying disease, previous pretransplant therapies, the age at HSCT, gender, total body irradiation, posttransplant derangement of immune system, and in the allogeneic setting, the presence of graft-versus-host disease requiring prolonged steroid treatment. Early identification of endocrine complications can greatly improve the quality of life of long-term survivors after HSCT. PMID:24883377

  14. Sublabial Autologous Ear Cartilage Grafting for Increasing the Nasolabial Angle

    PubMed Central

    Toncic, Dinko

    2016-01-01

    Background The loss of nasal tip support is caused by many factors and eventually results in the collapse and eventual dropping of the nasal tip. This reduces the nasolabial (NL) angle and negatively affects respiratory functions and one's appearance. Methods The aim of this retrospective study, which was conducted on 52 patients, was to present and popularize a simple and effective method for the reconstruction of a weakened columella by inserting an autologous ear cartilage graft using a sublabial approach. Results Of all the patients, three patients experienced transplant rejection. The period of follow-up observation was one to five years (mean, 27 months). The results were objectively evaluated by measuring the NL angle in standardized photos before and after the procedure at different time intervals over the follow-up period. We observed a significant increase of the NL angle (mean, 20°), and found these results to be durable over the long term. Of the 52 patients included in this study observed patients, three were dissatisfied (due to immediate infection and shifting of the strut), 28 were satisfied, and 21 were very satisfied. Conclusions The surgical method described here is simple and can be learned quickly. It has very good results with few complications, and is our method of choice for complex and serious cases seen in everyday rhinosurgical practice. PMID:26848445

  15. Older Patients with Myeloma Derive Similar Benefit from Autologous Transplantation

    PubMed Central

    Sharma, Manish; Zhang, Mei-Jie; Zhong, Xiaobo; Abidi, Muneer H.; Akpek, Görgün; Bacher, Ulrike; Callander, Natalie S.; Dispenzieri, Angela; Freytes, César O.; Fung, Henry C.; Gale, Robert Peter; Gasparetto, Cristina; Gibson, John; Holmberg, Leona A.; Kindwall-Keller, Tamila L.; Klumpp, Thomas R.; Krishnan, Amrita Y.; Landau, Heather J.; Lazarus, Hillard M.; Lonial, Sagar; Maiolino, Angelo; Marks, David I.; Mehta, Paulette; Med, Joseph R. Mikhael; Nishihori, Taiga; Olsson, Richard; Ramanathan, Muthalagu; Roy, Vivek; Savani, Bipin N.; Schouten, Harry C.; Scott, Emma; Tay, Jason; To, Luen Bik; Vesole, David H.; Vogl, Dan T.; Hari, Parameswaran

    2014-01-01

    Autologous hematopoietic cell transplantation (AHCT) for plasma cell myeloma is performed less often in people >70 years old than in people ≤70 years old. We analyzed 11,430 AHCT recipients for plasma cell myeloma prospectively reported to the Center for International Blood and Marrow Transplant Research between 2008 and 2011, representing the majority of US AHCT activity during this period. Survival (OS) was compared in 3 cohorts: ages 18 to 59 years (n = 5818), 60 to 69 years (n = 4666), and >70 years (n = 946). Median OS was not reached for any cohort. In multivariate analysis, increasing age was associated with mortality (P = .0006). Myeloma-specific mortality was similar among cohorts at 12%, indicating an age-related effect on nonmyeloma mortality. Analyses were performed in a representative subgroup comparing relapse rate, progression-free survival (PFS), and nonrelapse mortality (NRM). One-year NRM was 0% for age >70 years and 2% for other ages (P = not significant). The three-year relapse rate was 56% in age 18 to 59 years, 61% in age 60 to 69 years, and 63% age >70 (P = not significant). Three-year PFS was similar at 42% in age 18 to 59 years, 38% in age 60 to 69 years, and 33% in age >70 years (P = not significant). Postrelapse survival was significantly worse for the older cohort (P = .03). Older subjects selected for AHCT derived similar antimyeloma benefit without worse NRM, relapse rate, or PFS. PMID:25046833

  16. Quality Improvement Methodologies Increase Autologous Blood Product Administration

    PubMed Central

    Hodge, Ashley B.; Preston, Thomas J.; Fitch, Jill A.; Harrison, Sheilah K.; Hersey, Diane K.; Nicol, Kathleen K.; Naguib, Aymen N.; McConnell, Patrick I.; Galantowicz, Mark

    2014-01-01

    Abstract: Whole blood from the heart–lung (bypass) machine may be processed through a cell salvaging device (i.e., cell saver [CS]) and subsequently administered to the patient during cardiac surgery. It was determined at our institution that CS volume was being discarded. A multidisciplinary team consisting of anesthesiologists, perfusionists, intensive care physicians, quality improvement (QI) professionals, and bedside nurses met to determine the challenges surrounding autologous blood delivery in its entirety. A review of cardiac surgery patients’ charts (n = 21) was conducted for analysis of CS waste. After identification of practices that were leading to CS waste, interventions were designed and implemented. Fishbone diagram, key driver diagram, Plan–Do–Study–Act (PDSA) cycles, and data collection forms were used throughout this QI process to track and guide progress regarding CS waste. Of patients under 6 kg (n = 5), 80% had wasted CS blood before interventions, whereas those patients larger than 36 kg (n = 8) had 25% wasted CS before interventions. Seventy-five percent of patients under 6 kg who had wasted CS blood received packed red blood cell transfusions in the cardiothoracic intensive care unit within 24 hours of their operation. After data collection and didactic education sessions (PDSA Cycle I), CS blood volume waste was reduced to 5% in all patients. Identification and analysis of the root cause followed by implementation of education, training, and management of change (PDSA Cycle II) resulted in successful use of 100% of all CS blood volume. PMID:24783313

  17. First in Man: Sternal Reconstruction with Autologous Stem Cells.

    PubMed

    Khalpey, Zain; Marsh, Katherine M; Ferng, Alice; Riaz, Irbaz Bin; Hemphill, Courtney; Johnson, Kitsie; Oliva, Isabel; Friedman, Mark

    2015-01-01

    Sternal nonunion is associated with high morbidity and treated using rigid plate and screw fixation. This is the first reported example of successful sternal reconstruction using adipose-derived stromal vascular fraction (SVF) stem cells in addition to traditional techniques. Mesenchymal stem cells, one component of the SVF, play an important role in bone healing and were therefore used to promote remedial processes in a patient with sternal nonunion. A 3D printed model of the patient's sternum was used for preoperative planning of the plating. Intraoperatively, SVF was isolated using ultrasonic cavitation and previously planned sternal plating was completed. A total of 300 million cells were delivered via both local injection and intravenously before chest closure. The patient's pain dramatically decreased, commensurate with healed areas of nonunion by 3 months and maintained at 6 months postoperatively, supported by three-dimensional computed tomography imaging. Utilizing autologous stem cells from the SVF in conjunction with existing plating techniques may provide an optimal platform to stabilize the sternum and promote bone healing, although additional study is recommended. PMID:25914951

  18. Genome-wide expressions in autologous eutopic and ectopic endometrium of fertile women with endometriosis

    PubMed Central

    2012-01-01

    Background In order to obtain a lead of the pathophysiology of endometriosis, genome-wide expressional analyses of eutopic and ectopic endometrium have earlier been reported, however, the effects of stages of severity and phases of menstrual cycle on expressional profiles have not been examined. The effect of genetic heterogeneity and fertility history on transcriptional activity was also not considered. In the present study, a genome-wide expression analysis of autologous, paired eutopic and ectopic endometrial samples obtained from fertile women (n = 18) suffering from moderate (stage 3; n = 8) or severe (stage 4; n = 10) ovarian endometriosis during proliferative (n = 13) and secretory (n = 5) phases of menstrual cycle was performed. Methods Individual pure RNA samples were subjected to Agilent’s Whole Human Genome 44K microarray experiments. Microarray data were validated (P < 0.01) by estimating transcript copy numbers by performing real time RT-PCR of seven (7) arbitrarily selected genes in all samples. The data obtained were subjected to differential expression (DE) and differential co-expression (DC) analyses followed by networks and enrichment analysis, and gene set enrichment analysis (GSEA). The reproducibility of prediction based on GSEA implementation of DC results was assessed by examining the relative expressions of twenty eight (28) selected genes in RNA samples obtained from fresh pool of eutopic and ectopic samples from confirmed ovarian endometriosis patients with stages 3 and 4 (n = 4/each) during proliferative and secretory (n = 4/each) phases. Results Higher clustering effect of pairing (cluster distance, cd = 0.1) in samples from same individuals on expressional arrays among eutopic and ectopic samples was observed as compared to that of clinical stages of severity (cd = 0.5) and phases of menstrual cycle (cd = 0.6). Post hoc analysis revealed anomaly in the expressional profiles of several genes

  19. Use of autologous blood as part of the perfusate for cardiopulmonary bypass: a priming technique.

    PubMed

    Myers, G J; Legare, J F; Sullivan, J A; Leadon, R B; Johnstone, R; Swyer, W; Squires, C; Power, C; Hirsch, G M

    2002-05-01

    In an attempt to replace the oncotic and protein coating capabilities of serum albumin in the perfusate, we established a priming protocol that used autologous blood as part of the perfusate solution. Prior to March 1, 1999, our standard priming protocol was 1650 ml of crystalloid with 250 ml of 5% serum albumin and 5,000 units of heparin. After removing albumin from our prime, our standard protocol was altered to include 40 ml of the patient's autologous blood in 1,800 ml of crystalloid and 10,000 units of heparin. To determine the intraoperative effects of using albumin/crystalloid primes (Group A), autologous blood/crystalloid primes (Group B) and crystalloid primes (Group C), a total of 178 patients were sequentially evaluated. Intraoperative parameters evaluated were total protein (TP), colloid osmotic pressure (COP), platelets (Plts) and fluid requirements during cardiopulmonary bypass (CPB). During an overlapping 12-month period of time, 1,092 consecutive cardiac surgical cases using CPB (584 albumin prime; 508 autologous blood prime) were evaluated for clinical outcomes in terms of mortality and length of hospitalization. In addition, over a period of 15 months, 1,458 patients in both the autologous blood/crystalloid group and the crystalloid only group were evaluated for the incidence of high-pressure excursions (HPE) after going on bypass. Comparative reviews of TP, COP and Plts demonstrated no significant difference 10 min after the start of bypass between Groups A and B. However, in Group C, there was a statistically significant increase in the intraoperative fluid requirements during CPB, compared to both of the other groups. There was no significant difference in the incidence of HPE, with an occurrence of 1.04% in the crystalloid only group and 1.11% in the autologous blood/crystalloid group. Autologous blood perfusates were identical to albumin perfusates in their platelet protection and reduction of fluid shifts during the intraoperative period

  20. Transfer RNA-derived small RNAs in the cancer transcriptome.

    PubMed

    Green, Darrell; Fraser, William D; Dalmay, Tamas

    2016-06-01

    The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis. These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such disruption takes place. RNA silencing is a term used to describe the effects of post-transcriptional gene silencing mediated by a diverse set of small RNA molecules. Small RNAs are crucial for regulating gene expression and microguarding genome integrity. RNA silencing studies predominantly focus on small RNAs such as microRNAs, short-interfering RNAs and piwi-interacting RNAs. We describe an emerging renewal of interest in a 'larger' small RNA, the transfer RNA (tRNA). Precisely generated tRNA-derived small RNAs, named tRNA halves (tiRNAs) and tRNA fragments (tRFs), have been reported to be abundant with dysregulation associated with cancer. Transfection of tiRNAs inhibits protein translation by displacing eukaryotic initiation factors from messenger RNA (mRNA) and inaugurating stress granule formation. Knockdown of an overexpressed tRF inhibits cancer cell proliferation. Recovery of lacking tRFs prevents cancer metastasis. The dual oncogenic and tumour-suppressive role is typical of functional small RNAs. We review recent reports on tiRNA and tRF discovery and biogenesis, identification and analysis from next-generation sequencing data and a mechanistic animal study to demonstrate their physiological role in cancer biology. We propose tRNA-derived small RNA-mediated RNA silencing is an innate defence mechanism to prevent oncogenic translation. We expect that cancer cells are percipient to their ablated control of transcription and attempt to prevent loss of genome control

  1. Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent.

    PubMed

    Son, K K; Tkach, D; Patel, D H

    2000-09-29

    We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent. PMID:11018646

  2. Differential activation of RNA polymerase III-transcribed genes by the polyomavirus enhancer and the adenovirus E1A gene products.

    PubMed Central

    Berger, S L; Folk, W R

    1985-01-01

    We have compared the effect of the polyomavirus cis-acting transcriptional enhancer and the adenovirus trans-acting E1A gene on expression of RNA polymerase III-transcribed genes (the adenovirus VAI gene and a bacterial tRNA gene) using DNA transfection and transient expression assays. The polyomavirus enhancer has little effect upon transcription of the VAI gene by RNA polymerase III in any cell type tested (murine, hamster, or human). In contrast, expression of the E1A gene within adenovirus infected cells stimulates transcription of RNA polymerase III-transcribed genes from co-transfected DNAs. Human 293 cells, which constitutively produce adenovirus E1A gene products, also express high levels of RNA polymerase III transcripts from transfected DNAs. Images PMID:2987823

  3. Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase

    PubMed Central

    Gowher, Ali; Smirnov, Alexandre; Tarassov, Ivan; Entelis, Nina

    2013-01-01

    In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNALysCUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNALysCUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms. PMID:23799079

  4. Chitosan/siRNA nanoparticles encapsulated in PLGA nanofibers for siRNA delivery.

    PubMed

    Chen, Menglin; Gao, Shan; Dong, Mingdong; Song, Jie; Yang, Chuanxu; Howard, Kenneth Alan; Kjems, Jørgen; Besenbacher, Flemming

    2012-06-26

    Composite nanofibers of biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) encapsulating chitosan/siRNA nanoparticles (NPs) were prepared by electrospinning. Acidic/alkaline hydrolysis and a bulk/surface degradation mechanism were investigated in order to achieve an optimized release profile for prolonged and efficient gene silencing. Thermo-controlled AFM in situ imaging not only revealed the integrity of the encapsulated chitosan/siRNA polyplex but also shed light on the decreasing T(g) of PLGA on the fiber surfaces during release. A triphasic release profile based on bulk erosion was obtained at pH 7.4, while a triphasic release profile involving both surface erosion and bulk erosion was obtained at pH 5.5. A short alkaline pretreatment provided a homogeneous hydrolysis and consequently a nearly zero-order release profile. The interesting release profile was further investigated for siRNA transfection, where the encapsulated chitosan/siRNA NPs exhibited up to 50% EGFP gene silencing activity after 48 h post-transfection on H1299 cells. PMID:22621383

  5. Proteome alteration induced by hTERT transfection of human fibroblast cells

    PubMed Central

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

    2008-01-01

    Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase

  6. Evaluation of In Vivo Transfection Efficiency of Eudragit Coated Nanoparticles of Chitosan-DNA: A pH-sensitive System Prepared for Oral DNA Delivery

    PubMed Central

    Momenzadeh, Sedigheh; Sadeghi, Abdorrahim; Vatandoust, Nasimeh; Salehi, Rasoul

    2015-01-01

    Background: Success of any gene therapy protocol relies mostly on using an efficient carrier to direct nucleic acid to the place of action. The system should also have transfection ability at release site. Different routes are available for delivering genetic materials to the target organs, amongst them; oral delivery is particularly attractive for certain reasons. However, serious obstacles, like acidic environment of stomach and presence of protease and nuclease enzymes in gastrointestinal (GI) tract, make oral route a highly challenging option. Objectives: The present study suggests preparation of gene nanoparticles (NPs) of chitosan within a layer of Eudragit L100 for oral delivery of nucleic acid. The nanoparticles have some features both in size and polymer properties that can be penetrating enough to transfect epithelial layer cells of intestine and protect the entrapped materials against stomach harsh condition. Materials and Methods: In this experimental study, conducted in Iran, particles were prepared by coacervation technique followed by encapsulation of nanoparticle within a coat of Eudragit L100 using solvent evaporation technique. Formulation behavior was monitored both in vitro and in vivo. Stability of particle construction and release profile of DNA were examined at pH of ± 0.8 environ pKa of Eudragit. Size and zeta potential of particles were measured. To demonstrate transfection efficiency of the constructed carrier, reverse transcription polymerase chain reaction (RT-PCR) was carried out using human insulin specific primers on total RNA extracted from upper part of small intestine of 48-hour post-transfected rats (sampled by simple random selection, n = 3). Results: The mean size and zeta potential of particles were 300 ± 4 nm and 14 ± 0.5 mV, respectively. Encapsulation of this system was 89.6 ± 1.2%. DNA release from batches was less than 12% at pH = 5.2 and more than 60% at pH = 6.8 with significant difference of P < 0.05. RT-PCR product

  7. Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death.

    PubMed

    Wendt, G; Kemmel, V; Patte-Mensah, C; Uring-Lambert, B; Eckert, A; Schmitt, M J; Mensah-Nyagan, A G

    2014-03-28

    Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for

  8. Generation of human induced pluripotent stem cells using non-synthetic mRNA.

    PubMed

    Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

    2016-05-01

    Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. PMID:27064648

  9. Magnetic nanoparticle and magnetic field assisted siRNA delivery in vitro.

    PubMed

    Mykhaylyk, Olga; Sanchez-Antequera, Yolanda; Vlaskou, Dialechti; Cerda, Maria Belen; Bokharaei, Mehrdad; Hammerschmid, Edelburga; Anton, Martina; Plank, Christian

    2015-01-01

    This chapter describes how to design and conduct experiments to deliver siRNA to adherent cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles (MNPs). These magnetic complexes are targeted to the cell surface by the application of a gradient magnetic field. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected MNPs, phospholipids, and siRNAs are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres (microbubbles). Methods are described how to accomplish the synthesis of magnetic nanoparticles for magnetofection and how to test the association of siRNA with the magnetic components of the transfection vector. A simple method is described to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Procedures are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA as well as magnetic microbubbles for magnetofection and downregulation of the target gene expression analysis with account for the toxicity determined using an MTT-based respiration activity test. A modification of the magnetic transfection triplexes with INF-7, fusogenic peptide, is described resulting in reporter gene silencing improvement in HeLa, Caco-2, and ARPE-19 cells. The methods described can also be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transfection by magnetofection in any cell type. PMID:25319646

  10. Hyaluronidase and collagenase increase the transfection efficiency of gene electrotransfer in various murine tumors.

    PubMed

    Cemazar, Maja; Golzio, Muriel; Sersa, Gregor; Escoffre, Jean-Michel; Coer, Andrej; Vidic, Suzana; Teissie, Justin

    2012-01-01

    One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy. PMID:21797718

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

    PubMed Central

    2010-01-01

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

  12. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  13. Influence of needle gauge on in vivo ultrasound and microbubble-mediated gene transfection.

    PubMed

    Browning, Richard J; Mulvana, Helen; Tang, Mengxing; Hajnal, Jo V; Wells, Dominic J; Eckersley, Robert J

    2011-09-01

    Ultrasound and microbubble-mediated gene transfection are potential tools for safe, site-selective gene therapy. However, preclinical trials have demonstrated a low transfection efficiency that has hindered the progression of the technique to clinical application. In this paper it is shown that simple changes to the method of intravenous injection can lead to an increase in transfection efficiency when using 6-MHz diagnostic ultrasound and the ultrasound contrast agent, SonoVue. By using needles of progressively smaller gauge, i.e., larger internal diameter (ID), from 29 G (ID 0.184 mm) to 25 G (ID 0.31 mm), the transfection of a luciferase plasmid (pGL4.13) was significantly increased threefold in heart-targeted female CD1 mice. In vitro work indicated that the concentration and size distribution of SonoVue were affected by increasing needle gauge. These results suggest that the process of systemic delivery alters the bubble population and adversely affects transfection. This is exacerbated by using high-gauge needles. These findings demonstrate that the needle with the largest possible ID should be used for systemic delivery of microbubbles and genetic material. PMID:21741156

  14. Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(l-lysine).

    PubMed

    Liu, Shuai; Yang, Jixiang; Ren, Hongqi; O'Keeffe-Ahern, Jonathan; Zhou, Dezhong; Zhou, Hao; Chen, Jiatong; Guo, Tianying

    2016-03-01

    Natural polycations, such as poly(l-lysine) (PLL) and chitosan (CS), have inherent superiority as non-viral vectors due to their unparalleled biocompatibility and biodegradability. However, the application was constrained by poor transfection efficiency and safety concerns. Since previous modification strategies greatly weakened the inherent advantages of natural polycations, developing a strategy for functional group introduction with broad applicability to enhance the transfection efficiency of natural polycations without compromising their