scholarly journals 46 EFFICIENT TRANSFECTION OF PLASMID DNA INTO CELLS FOR USE AS NUCLEAR DONORS

2005 ◽  
Vol 17 (2) ◽  
pp. 172
Author(s):  
S.-L. Lee ◽  
S.-A. Ock ◽  
H.-J. Song ◽  
B. Mohana Kumar ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
South Korea ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cumulus Cells ◽  
Cell Types ◽  
Development Project ◽  
Exogenous Dna ◽  
Transfected Cells ◽  
Fetal Fibroblasts

Somatic cell nuclear transfer (SCNT) has the potential to significantly improve the production of valuable livestock that produce recombinant proteins, such as pharmaceutical proteins for human disease or biomaterials for medical use. The success of this potential depends on efficient and optimized protocols for introducing exogenous DNA into cells. In this study, we compared two methods of transfection, Effectene (Qiagen, Inc., Valencia, CA, USA) and electroporation. Plasmid DNA (pEGFF-N1, Clontech, Seoul, Korea) was transfected into fetal fibroblasts (FFB), cumulus cells (CUC), and adult ear skin cells (ESC). Transfection efficiency, chromosome normality, gene expression, and apoptosis were assessed. Cells cultured in α-modified Eagle's medium (α-MEM; BioWhittaker, Walkersville, MD, USA) + 10% FBS were transfected with pEGFP-N1. For electroporation, cells (5 × 106 cells/mL) were mixed in 300 μL perrim buffer (75% Cytosalts with 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 5 mM MgCl2, and 25% α-MEM) + 15 μg pEGFP-N1, and subjected to two pulses of 0.38 kV and 400 μF delivered by Gene Pulser (Bio-Rad; BMS, Ltd., Seoul, South Korea). For Effectene transfection, the procedure suggested by the manufacture was followed. Transfected cells were selected with 600 μg/mL G418 (Gibco; KDR Biotech Co., Ltd., Seoul, South Korea) and cultured at 39°C, 5% CO2 in air. Assessments of EGFP transfected cells by green fluorescence was carried out under an inverted epifluorescence microscope (Nicon, Kanagawa, Japan) equipped with a filter for FITC (excitation maximum = 488 nm; emission maximum = 507 nm). Differences among the groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Most cells (>80%) in confluence were at G0/G1 phase, and transfection of the gene into all three cell types did not affect the incidence of chromosomal abnormality or change morphology. In addition, the rates of apoptosis assessed by TUNEL did not differ in all three cell types by either method of transfection at different cell passages. However, the efficiency of gene transfection into FFB by Effectene reagent (14.2 ± 1.7%) was significantly (P < 0.05) higher than that by electroporation (5.1 ± 1.0%). Among the three type cells, the efficiency of gene transfection by Effectene and electroporation of FFB (14.2 ± 1.7 and 5.1 ± 1.0%, respectively) was significantly (P < 0.05) higher than those of CUC and ESC (9.4 ± 1.5 and 3.3 ± 0.8; 8.8 ± 0.7 and 2.1 ± 0.4%, respectively). In conclusion, although there were no differences in the alteration of chromosomes, cell morphology, and apoptosis among three cell types transfected with or without plasmid DNA, FFB is the most effective cell type to be transfected. Effectene is superior to other currently available methods for introducing plasmid DNA into a variety of cells. The high level of transfection achieved by Effectene will encourage its use as a tool for producing transgenic embryos and animals by SCNT. This work was supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010.

Free radical-mediated transgene inactivation of macrophages by endotoxin

2000 ◽  
Vol 279 (5) ◽  
pp. L878-L883 ◽  
Author(s):  
Sujatha Dokka ◽  
David Toledo ◽  
Liying Wang ◽  
Xianglin Shi ◽  
Chuanshu Huang ◽  
...  
Keyword(s):  
Free Radical ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cationic Liposome ◽  
Sodium Formate ◽  
Cell Types ◽  
Polymyxin B ◽  
Free Radical Scavengers ◽  
Metal Catalyzed

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N- t-butyl-α-phenylnitrone, the H2O2 scavenger catalase, and the ·OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O2 − scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that ·OH formed by H2O2-dependent, metal-catalyzed Fenton reaction play a major role in this process.

scholarly journals Dual delivery of nucleic acids and PEGylated-bisphosphonates via calcium phosphate nanoparticles

2019 ◽  
Author(s):  
Sofia Bisso ◽  
Simona Mura ◽  
Bastien Castagner ◽  
Patrick Couvreur ◽  
Jean-Christophe Leroux
Keyword(s):  
Calcium Phosphate ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Physical Stability ◽  
Entrapment Efficiency ◽  
Endosomal Escape ◽  
Particle Uptake ◽  
Success Stories

AbstractDespite many years of research and a few success stories with gene therapeutics, efficient and safe DNA delivery remains a major bottleneck for the clinical translation of gene-based therapies. Gene transfection with calcium phosphate (CaP) nanoparticles brings the advantages of low toxicity, high DNA entrapment efficiency and good endosomal escape properties. The macroscale aggregation of CaP nanoparticles can be easily prevented through surface coating with bisphosphonate conjugates. Bisphosphonates, such as alendronate, recently showed promising anticancer effects. However, their poor cellular permeability and preferential bone accumulation hamper their full application in chemotherapy. Here, we investigated the dual delivery of plasmid DNA and alendronate using CaP nanoparticles, with the goal to facilitate cellular internalization of both compounds and potentially achieve a combined pharmacological effect on the same or different cell lines. A pH-sensitive poly(ethylene glycol)-alendronate conjugate was synthetized and used to formulate stable plasmid DNA-loaded CaP nanoparticles. These particles displayed good transfection efficiency in cancer cells and a strong cytotoxic effect on macrophages. The in vivo transfection efficiency, however, remained low, calling for an improvement of the system, possibly with respect to the extent of particle uptake and their physical stability.Graphical abstract

scholarly journals Cellular Injury of Cardiomyocytes during Hepatocyte Growth Factor Gene Transfection with Ultrasound-Triggered Bubble Liposome Destruction

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Kazuo Komamura ◽  
Rie Tatsumi ◽  
Yuko Tsujita-Kuroda ◽  
Takatoshi Onoe ◽  
Kunio Matsumoto ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cellular Injury ◽  
Factor Gene ◽  
Trade Offs ◽  
Growth Factor Gene ◽  
Low Concentrations ◽  
Hepatocyte Growth ◽  
Cultured Cardiomyocytes

We transfected naked HGF plasmid DNA into cultured cardiomyocytes using a sonoporation method consisting of ultrasound-triggered bubble liposome destruction. We examined the effects on transfection efficiency of three concentrations of bubble liposome (1×106, 1×107, 1×108/mL), three concentrations of HGF DNA (60, 120, 180 μg/mL), two insonification times (30, 60 sec), and three incubation times (15, 60, 120 min). We found that low concentrations of bubble liposome and low concentrations of DNA provided the largest amount of the HGF protein expression by the sonoporated cardiomyocytes. Variation of insonification and incubation times did not affect the amount of product. Following insonification, cardiomyocytes showed cellular injury, as determined by a dye exclusion test. The extent of injury was most severe with the highest concentration of bubble liposome. In conclusion, there are some trade-offs between gene transfection efficiency and cellular injury using ultrasound-triggered bubble liposome destruction as a method for gene transfection.

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 151-158 ◽  
Author(s):  
Rita P. Cervera ◽  
Fernando García-Ximénez
Keyword(s):  
Technical Efficiency ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cell Types ◽  
Cell Type ◽  
Fetal Fibroblasts ◽  
Somatic Nuclear Transfer ◽  
Metaphase Ii Oocytes

The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion rate was obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.

scholarly journals Nonhomologous End-Joining Ligation Transfers DNA Regulatory Elements between Cointroduced Plasmids

2004 ◽  
Vol 24 (19) ◽  
pp. 8323-8331 ◽  
Author(s):  
Toshio Ishikawa ◽  
Eun Jig Lee ◽  
J. Larry Jameson
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Molecules ◽  
End Joining ◽  
Exogenous Dna ◽  
Strand Breaks

ABSTRACT Cointroduction of plasmids into mammalian cells is commonly used to investigate transcription factor regulation of reporter genes or to normalize transfection efficiency. We report here that cotransfected DNA molecules commonly transfer enhancer elements from one plasmid to another. Using separate Renilla or Firefly luciferase reporters, we found that an estrogen response element (ERE) originally linked to one of the reporters stimulated expression of the non-ERE-containing reporter. Similar enhancer transfer was seen with the cytomegalovirus enhancer. This enhancer transfer effect was not seen when cells were transfected separately with the reporters and the extracts were then combined before luciferase assays. The degree of enhancer transfer increased with transfected plasmid concentration and was greater when linearized rather than circular plasmid DNA was used. We hypothesized that double-strand breaks and heteroligation of cointroduced DNA molecules mediated the transfer of regulatory elements from one molecule to another. PCR of transfected plasmid DNA confirmed nonhomologous end-joining (NHEJ) ligation of DNA fragments originally present in separate plasmids. The NHEJ reaction was enhanced by UV light treatment to introduce double-strand breaks, and it was greater after liposome-mediated transfection than after calcium-phosphate-mediated transfection. NHEJ also occurred after adenoviral transfer of DNA into cells. We conclude that NHEJ mediates the transfer of regulatory DNA elements among cointroduced DNA molecules. These findings indicate the need for caution when interpreting results of transfection experiments containing more than one plasmid and suggest a mechanism whereby viruses or other exogenous DNA might recombine to activate unrelated genes.

Size effect of complexed plasmid DNA to gene transfection efficiency of microbubble-mediated sonoporation

2013 ◽  
Vol 133 (5) ◽  
pp. 3495-3495
Author(s):  
Yoichiro Matsumoto ◽  
Yiwei Zhang ◽  
Takashi Azuma ◽  
Kiyoshi Yoshinaka ◽  
Kensuke Osada ◽  
...  
Keyword(s):  
Size Effect ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection

scholarly journals Preparation and Characterization of Folate-Targeted Fe3O4 Nanoparticle Codelivering Cisplatin and TFPI-2 Plasmid DNA for Nasopharyngeal Carcinoma Therapy

2017 ◽  
Vol 2017 ◽  
pp. 1-10
Author(s):  
Juan Zhang ◽  
Huanhuan Weng ◽  
Xiangwan Miao ◽  
Quanming Li ◽  
Siqi Wang ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Fluorescence Analysis ◽  
Average Diameter ◽  
Transmission Electron Microscopy Image ◽  
Proton Nuclear Magnetic Resonance ◽  
Targeting Therapy ◽  
The Core

A novel folate (FA) receptor-targeted superparamagnetic Fe3O4 nanoparticles (SPIONs) codelivering cisplatin (CDDP) and tissue factor pathway inhibitor-2 (TFPI-2) plasmid DNA (pDNA) was constructed. The core shell nanocomposites (FA-PEG-PEI@SPION-CDDP-TFPI-2) were composed of superparamagnetic Fe3O4 core that binds CDDP and TFPI-2 shell that combines with folate-polyethylene glycol-polyethyleneimine (FA-PEG-PEI) via electrostatic interaction. The shell containing FA-PEG-PEI and TFPI-2 plasmid was synthesized through amidation reaction and electrostatic adsorption and the core containing SPION-CDDP was modified by aldehyde sodium alginate. Proton nuclear magnetic resonance and Fourier transform infrared spectra on FA-PEG-PEI polymers showed characteristic peaks of various metabolites in corresponding frequency. Transmission electron microscopy image of FA-PEG-PEI@SPION-CDDP-TFPI-2 nanoparticles demonstrated a near-monodisperse spherical morphology, while dynamic light scattering studies indicated an intensity-average diameter of 149.5 nm. Zeta potential was 14.89 ± 1.83 mv and the final concentration of loaded CDDP was 100 ug/ml. Gel electrophoresis data showed that the nanocomposite would protect TFPI-2 pDNA from being digested by DNases. Compared with CNE-2 cells, the good targetability and better gene transfection efficiency (57.9%) were detected by Prussian blue iron stain and fluorescence analysis in HNE-1 cells in vitro. The results suggested the potential application of FA-PEG-PEI@SPION-CDDP-TFPI-2 as a multifunctional anticancer nanomedicine on targeting therapy for FR positive NPC.

scholarly journals Size effect of complexed plasmid DNA to gene transfection efficiency of microbubble-mediated sonoporation

2013 ◽  
Author(s):  
Yoichiro Matsumoto ◽  
Yiwei Zhang ◽  
Takashi Azuma ◽  
Kiyoshi Yoshinaka ◽  
Kensuke Osada ◽  
...  
Keyword(s):  
Size Effect ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection

197 INFLUENCE OF PLASMID CONSTRUCTION AND CELL TYPE ON THE GENERATION OF GENETICALLY MODIFIED CELL LINES IN CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 189
Author(s):  
A. G. Curcio ◽  
F. F. Bressan ◽  
K. S. Viana ◽  
A. F. L. Rios ◽  
F. V. Meirelles ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Production Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Specific Cell ◽  
Cell Type ◽  
Transfected Cells ◽  
Plasmid Construction ◽  
Fetal Fibroblasts ◽  
Adult Fibroblasts

Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN2 plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP+ cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student–Newman–Keuls test (SNK; P < 0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65, CC: 3.9 ± 1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542 ± 497.09; AF: 9367.5 ± 3490.9, CC: 3496 ± 2638.92). The pEGFPN2 plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 – FF: 4.9 ± 0.14 and 206.47 ± 755; AF: 760 and 2.4 ± 0.70 ± 330.92; CC: 3.9 ± 1.97, and 1418 ± 36.06, respectively; control – FF: 0.15 ± 0.07 and 249 ± 6 : 36; AF: 0.15 ± 0.07 588 ± 213.54, and CC 0.05 ± 0 214 ± 0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.

Developmental competence and gene expression in preimplantation bovine embryos derived from somatic cell nuclear transfer using different donor cells

Zygote ◽  
2005 ◽  
Vol 13 (3) ◽  
pp. 187-195 ◽  
Author(s):  
Goo Jang ◽  
Hyun Yong Jeon ◽  
Kyung Hee Ko ◽  
Hee Jung Park ◽  
Sung Keun Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Cell Types ◽  
Developmental Competence ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Different Cell Types

This study compared the developmental competence of somatic cell nuclear transfer (SCNT) embryos reconstructed with different donor cells and analysed gene expression in the resulting embryos. Bovine fetal/adult ear fibroblasts and cumulus cells were used as donor cells and the developmental competence of the reconstructed embryos was monitored. The cell number and allocation in blastocysts were determined by differential staining. The Bax, E-cad, IF-tau, Hsp (heat shock protein) 70, Igf2r (insulin-like growth factor 2 receptor), DNMT (DNA methyltransferase) 1 and Mash (mammalian achaete-scute homologue) 2 genes were selected for gene expression analysis. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by semiquantitative reverse transcription-polymerase chain reaction. In experiment 1, development of SCNT preimplantation embryos and the cell numbers of inner cell masses and trophoblasts were not different among SCNT embryos derived from different cell types. In experiment 2, the relative expression of GAPDH and Hsp 70 transcripts was similar in all embryos. The expression of Bax, Igf2r and Mash2 transcripts was significantly increased in SCNT embryos reconstructed with adult fibroblasts. The E-cad transcript levels were reduced in SCNT embryos reconstructed with fetal fibroblasts. Relative abundance of DNMT1 in SCNT embryos derived from fetal fibroblasts was increased, and IF-tau expression in SCNT embryos derived from cumulus cells was increased. In conclusion, depending on the type of donor cells, preimplantation SCNT embryos displayed marked differences in gene expression. This may affect the developmental competence of SCNT embryos reconstructed with different cell types after implantation or during fetal growth in vivo.

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