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741 results for “transfection”
Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells
<p>Raw data of the publication "Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells".</p> <p>Abstract: Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal<br> neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor<br> degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell<br> growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated,<br> often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are<br> costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene<br> therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor<br> (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression<br> of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system<br> that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a<br> low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated<br> the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as<br> primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed<br> by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected<br> in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination<br> with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while<br> ensuring high transfection efficiency and long-term transgene expression in RPE cells.</p>
Protocol for transfection by microinjection into the eggs of the parasite vector snail Biomphalaria glabrata
<p><strong>1. Egg production</strong></p> <p>Place about 30 adult snails (10 mm diameter) into a 5.5-liter water tank. Place a piece of polystyrene of (3 x 3 cm) in each tank. There is the preferred support of <em>Biomphalaria glabrata</em> for laying its eggs. The snails are fed <em>ad libitum</em> with green lettuce leaves, they can also be fed with dry spirulina to boost reproduction. Maintain water at a temperature of 25 degrees Celsius.</p> <p><strong>2. Egg collection</strong></p> <p>Gently pick up several egg layers from the polystyrene with soft holding forceps and place the eggs into a petri dish with natural mineral water (e.g. Volvic) to prevent them from drying out.</p> <p>Start sorting the eggs under the stereoscopic microscope to choose only the gastrula stage and place them into another petri dish with natural mineral water.</p> <p> </p> <p><strong>3. Preparation of the transfection solution</strong></p> <p><strong>Material:</strong></p> <p>a. <em>in vivo</em> JetPEI transfection reagent</p> <p>b. 10% glucose solution</p> <p>c. 5% glucose solution</p> <p>d. Plasmids (dCas9-SunTag-BFP and scFv-DNMT3A-GFP)</p> <p>e. 0.2 ml microtubes</p> <p>f. P10 and P200 pipettes</p> <p>g. P10 and P200 pipette tips</p> <p>h. Permanent marker</p> <p>The glucose solution and the <em>in vivo</em> jetPEI transfection reagent are equilibrated at room temperature. </p> <p>Prepare 21 µl of each plasmid at a concentration of 78 and 88 ng / µl respectively (for a total volume of 42 µl =equals 3.5 µg of DNA) add the plasmid DNA to a 0.2 ml tube (labeled as Tube A) and mix with 21 µl of 10% glucose solution. </p> <p>In another microtube (labeled as Tube B), add 21 μl of 5% glucose solution and 1 μl of <em>in vivo</em> jetPEI. </p> <p> Prepare a third tube (labeled as Tube C) with 21 µl of 5% glucose solution and 0.5 µl of <em>in vivo</em> jetPEI to inject into embryos that will serve as controls. </p> <p>Leave the solutions at room temperature while you prepare the microinjection station. </p> <p> </p> <p> </p> <p><strong>4. Preparation of the micro-injection station</strong></p> <p><strong>Material:</strong></p> <p>a. Pre-pulled glass micropipettes (1mm diameter)</p> <p>b. Watch glass</p> <p>c. Modeling clay</p> <p>d. 35 mm and 90 mm petri dishes</p> <p>e. Mineral oil (M5904, SIGMA)</p> <p>f. Wash bottle with natural mineral water (Volvic)</p> <p>g. 0.2 ml microtubes</p> <p>h. 12-well cell culture plate</p> <p>i. Fine brush</p> <p>j. Phenol red solution</p> <p>k. Pasteur pipette or dropper</p> <p>l. Dissection forceps</p> <p>m. Soft holding forceps</p> <p>n. Snail eggs in the gastrula stage</p> <p>o. Drummond Scientific Nanoject III Programmable Nanoliter Injector</p> <p> </p> <p>Take a pre-pulled glass micropipette and cut it with a scalpel to have a ~ 0.2 mm tip slightly beveled if possible.</p> <p>Before attaching the micropipette to the programmable nanoliter injector, fill it with mineral oil. If this step is not done, the injector will not work properly. This can be done with a filling needle </p> <p>attached to a hamilton syringe of 10 microliters.</p> <p>When the micropipette is filled with oil, it must be fixed on the injector. For this it is necessary to: </p> <p>Slide the chuck and collet onto the glass micropipette, then slide the black O-ring with the seal onto the wire plunger </p> <p>With the micropipette attached to the injector, press the [EMPTY] icon until the plunger is fully extended. This step can be done with the footswitch by pressing once [EMPTY] then [STOP] and then proceeding [EMPTY] with the foot switch. A single beep is emitted when the plunger is fully extended.</p> <p>Fill the micropipette with 3 µl of the control solution or the transfection solution by placing the glass micropipette tip in a 0.2 ml tube with the solution to be injected and pressing the [FILL] icon. It is desirable to fill it at a slow rate, by pressing the [FILL] icon for a few seconds, then the [STOP] icon to allow the sample to equilibrate before pressing again the '[FILL] icon.</p> <p>Note: The piston continues to extend or retract until the [STOP] icon is pressed, or until the fully extended or fully retracted position is reached.</p> <p> </p> <p>5. Microinjection</p> <p>Place a watch glass into a 35mm petri dish and secure it on one side with modeling clay to form a slope. Use soft handling forceps to transfer an egg mass and lay it on the slope side of the watch glass so that the egg mass is in a sloping position.</p> <p>Remove excess water from the eggs with absorbent paper. Rehydrate if necessary with a fine brush to improve the visibility of the embryos. To inject the sample, return to the operating mode screen by pressing the [EXIT] icon, then select the injection mode by pressing the [INJECT] icon. Set the injection volume to 30nL and the flow rate to 20nL per second using the icons [+] and [-] respectively. Press the [INJECT] icon to inject the sample. </p> <p>Inject 30nL of the microinjection solution into each egg. Place the microinjected egg masses in a 12-well cell culture plate and note with a marker whether they were microinjected with the control solution or with the solution containing the plasmids.</p> <p>We colored the injection solution with red phenol to facilitate the visibility in this video.</p> <p><strong>Monitor the expression of the plasmids</strong></p> <p>Monitor the plasmids expression 72 h after microinjection in a contrast / fluorescent microscope or in a fluorescent stereo microscope. Then sort the fluorescent snails and perform a second micro-injection with a solution containing 10 µl of single guide RNA (at a concentration of 2ng / µl), add 0.5 µl of <em>in vivo</em> jetPEI reagent and 10 µl of 5% glucose solution. 3 days after the second microinjection, collect the hatched snails in a 1.5 ml tube containing 25 µl of lysis buffer for DNA and RNA purification.</p> <p>In this photo produced under a confocal microscope we washed a veliger larva in PBS solution, then we fixed it with 4% paraformaldehyde solution and then we placed it in a slide with two drops of the Dako fluorescence mounting medium. </p> <p>96 after the transfection we can observe the expression of the green fluorescent protein, the blue fluorescent protein and the co-localization of both proteins. </p> <p>This protocol is used to perform DNA methylation changes in a target gene. This transfection protocol can be used with other plasmids, with small interfering RNAs, or with messenger RNAs.</p> <p>Produced at IHPE (http://ihpe.univ-perp.fr)</p>
Supplementing data and code for: "Correlation of mRNA delivery timing and protein expression in lipid-based transfection"
<p>Supplementary data and code for Reiser <em>et al.</em>: Correlation of mRNA delivery timing and protein expression in lipid-based transfection. 2019, <a href="https://doi.org/10.1093/intbio/zyz030">doi:10.1093/intbio/zyz030</a>.</p> <p>See `README.pdf` for further details.</p>
Production and purification of receptor-binding domain (RBD) of the Spike protein from a transiently transfected mammalian cell
<p> </p> <p>Production and purification of recombinant receptor-binding domain (RBD) of the Spike protein from a transiently transfected EXPI293 mammalian cell .</p>
Core-shell structured chitosan-polyethylenimine nanoparticles for gene delivery: Improved stability, cellular uptake, and transfection efficiency
<p>Gene therapy has emerged as a promising treatment option for various acquired and inherited diseases. The delivery of nucleic acids relies on so-called vectors that condense and encapsulate their cargo, generating stable nano-sized particles. Especially non-viral gene delivery systems are of increasing interest. However, accomplishing therapeutic levels of transgene expression and limited tolerability of these systems remain a challenge. Therefore, we investigate in the present study the improvement of nucleic acid delivery using depolymerized chitosan – polyethylenimine DNA core complexes (dCS-PEI/DNA). These core complexes are further entrapped into a variety of dCS-based shells, functionalized with poly(ethylene glycol) (PEG) spacers conjugated to ionic moieties (amino or carboxylate groups) and cell penetrating peptides. This modular approach allowed to evaluate the effect of the shell functional components on the physico-chemical particle characteristics and biological effects <em>in vitro</em>. The optimized ternary complex combines a core-dCS-LPEI/DNA complex with a shell consisting of dCS-PEG-COOH, which resulted in improved encapsulation of nucleic acid, accelerated cellular uptake, enhanced transfection efficiency, and superior transfection potency in human hepatoma HuH-7 cells and mouse primary hepatocytes. Effects on transgene expression are confirmed <em>in vivo</em> in wild-type mice following retrograde intrabiliary infusion. After administration to mice of only 100 ng complexed nanovector DNA, ternary complexes induce a high reporter gene signal for three days. We conclude that ternary core-shell structured particles comprising functionalized chitosan are a promising gene delivery technology for both <em>in vitro</em> as well as <em>in vivo </em>applications. The modular design will facilitate the development of chemically modified derivatives.</p>
ROS-specific Huntingtin Interactions: Comparing transfection methods for inducible expression of huntingtin-specific chromobody
<p>Optimization step in the development of an inducible system expressing YFP-tagged huntingtin-specific intrabodies for stable transfection in TruHD fibroblasts.</p>
Optimising C2C12 muscle myoblast transfection with polyethylenimine (PEI) or Lipofectamine-2000
<p>Optimisation of DNA transfection of the C2C12 muscle myoblast cell line, comparing use of polyethylenimine (PEI) and Lipofectamine-2000 with varying treatment conditions. </p>
Determining the transfectability of different patient-derived cell lines
<p>Determining whether the following patient-derived cell lines can be transfected efficiently using Lipofectamine 2000</p> <p>C2C12</p> <p>HSJD-DIPG-008</p> <p>HSJD-DIPG-011</p> <p>HSJD-DIPG-012</p> <p>HSJD-DIPG-013</p> <p>SU-DIPG-IV</p> <p>HSJD-GBM-002</p> <p>HSJD-DIPG-007</p> <p> </p>
A Phase 1, First Time in Human (FTIH) Study to Evaluate GSK3352589, a REarranged During Transfection (RET) Growth Factor Receptor Tyrosine Kinase Inhibitor, in Healthy Volunteers
ClinicalTrials.gov study NCT03154086. IPD Sharing: NO. Countries: 1. Publications: 1.
A Clinical Trial in Healthy, HIV-1-Uninfected Adult Participants to Compare the Safety, Tolerability and Immunogenicity of CH505TF gp120 Produced From Stably Transfected Cells to CH505TF gp120 Produce
ClinicalTrials.gov study NCT03856996. IPD Sharing: Not stated. Countries: 1. Publications: 2.
Tumor growth kinetics of human MDA-MB-231 cells transfected with dTomato lentivirus
<p>Tumor growth data involve human MDA-MB-231 cells stably transfected with dTomato lentivirus.</p> <p>Animals were orthotopically implanted (80,000 cells at injection) into the mammary fat pads of 6-week-old female nude mice.</p> <p>Tumor size was monitored regularly with fluorescence imaging. The data comprised a total of 64 observations.</p> <p> </p> <p>In the file, the columns correspond to:</p> <ul> <li>ID: identifier of the animal</li> <li>Time: day of the tumor measurement after implantation</li> <li>Observation: tumor measurement (in phot./s)</li> </ul> <p> </p> <p><strong>Please cite: </strong>Vaghi C, Rodallec A, Fanciullino R, Ciccolini J, Mochel JP, et al. (2020) Population modeling of tumor growth curves and the reduced Gompertz model improve prediction of the age of experimental tumors, PLoS Comput Biol, 16, p. e1007178. <a href="https://doi.org/10.1371/journal.pcbi.1007178">https://doi.org/10.1371/journal.pcbi.1007178</a></p>
Systemic optimization of gene electrotransfer protocol using hard-to-transfect UT-7 cell line as a model
<p><strong>Externally hosted supplementary file. </strong>Table S1: Table results of one-way ANOVA, followed by a post-hoc Tukey multiple comparison test. <em>P</em><0.05 value was considered statistically significant. Significant differences are marked in red. Table S2: Table results of one-way ANOVA, followed by a post-hoc Tukey multiple comparison test. <em>P</em><0.05 value was considered statistically significant. Significant differences are marked in red. Table S3: Table results of one-way ANOVA, followed by a post-hoc Tukey multiple comparison test. <em>P</em><0.05 value was considered statistically significant. Significant differences are marked in red.</p>
A Transfection-Free Approach of Gene Editing via a gold-based nanoformulation of the Cas9 protein
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Transfection of NSD3-tareting siRNA in H1299 Cells
<p><strong>SGC Open Notebook Project to Characterize the HMTase NSD3</strong></p> <p><strong>Exp019 Objective: </strong> I have identified a putative phenotype in H1299 cells upon overexpression of the NSD3 short<br> isoform. I am also interested in any phenotype resulting from decreased amounts of NSD3 in cells. To<br> do so, I am first testing conditions for RNAi-mediated knockdown of NSD3 in H1299 cells by treating cells<br> with several concentrations of siRNA and evaluating knockdown by western blotting</p>
Immunotherapy of Melanoma With Tumor Antigen RNA and Small Inhibitory RNA Transfected Autologous Dendritic Cells
ClinicalTrials.gov study NCT00672542. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Peptide-pulsed vs. RNA-transfected Dendritic Cell Vaccines in Melanoma Patients
ClinicalTrials.gov study NCT00243529. IPD Sharing: Not stated. Countries: 1. Publications: 7.
NADPH Oxidase Correction in mRNA-transfected Granulocyte-enriched Cells in Chronic Granulomatous Disease (CGD)
ClinicalTrials.gov study NCT05189925. IPD Sharing: Not stated. Countries: 1. Publications: 3.
Magnetic transfection with superparamagnetic chitosan-loaded IGFBP5 nanoparticles and their in vitro biosafety
<p><strong>Purpose: </strong>To study the application of superparamagnetic chitosan nanoparticles (SPCIONPs) as gene vectors using a magnetic transfection system for the targeted treatment of lung metastasis of osteosarcoma.</p> <p><strong>Methods: </strong>The superparamagnetic chitosan nanoparticles were characterized by Transmission Electron Microscopy, Fourier Transform Infrared spectrometry, a Superconducting Quantum Interference Device and Atomic Force Microscopy. Their biosafety was determined by cell counting kit-8 (CCK8) and live-dead staining assays. In vitro transfection was detected by laser confocal microscopy.</p> <p><strong>Results:</strong> SPCIONPs, which can bind closely to plasmids and protect them from DNA enzyme degradation, were prepared with an average particle size of 95.60 nm and zeta potential of 11 mV. The results of the CCK8 and live-dead staining assays showed that superparamagnetic chitosan nanoparticles loaded with Insulin-like growth factor-binding protein 5 (SPCIONPs/pIGFBP5) induced no significant cytotoxicity compared to the control group. The in vitro transfection result suggested that pIGFBP5 emitted a greater amount of red fluorescence in the SPCIONPs/pIGFBP5 group than that in the chitosan-loaded IGFBP5 (CS/pIGFBP5) group.</p> <p><strong>Conclusion: </strong>The prepared SPCIONPs had good biosafety and could be effectively used to transfer pIGFBP5 into 143B cells, and they thus have good application prospects for the treatment of lung metastasis of osteosarcoma.</p>
A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing
<p>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.</p>
Detection of SARS-CoV-2 E and N mRNA at 12 h after co-transfection with SARS-CoV-2 E and N plasmids.
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