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Artykuły w czasopismach na temat „Transfection”

Autor: Grafiati

Data publikacji: 4 czerwca 2021

Data aktualizacji: 30 lipca 2024

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1

Toth, Maria, Manuel Reithofer, Gregory Dutra, Patricia Pereira Aguilar, Astrid Dürauer i Reingard Grabherr. "Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells". Viruses 16, nr 3 (10.03.2024): 426. http://dx.doi.org/10.3390/v16030426.

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(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

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2

Cheow, Pheik-Sheen, Tiong Kit Tan, Adelene Ai-Lian Song, Khatijah Yusoff i Suet Lin Chia. "An improved method for the rescue of recombinant Newcastle disease virus". BioTechniques 68, nr 2 (luty 2020): 96–100. http://dx.doi.org/10.2144/btn-2019-0110.

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Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.

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Zhang, Jianxiong, Yawei Hu, Xiaoqing Wang, Peng Liu i Xiaofang Chen. "High-Throughput Platform for Efficient Chemical Transfection, Virus Packaging, and Transduction". Micromachines 10, nr 6 (10.06.2019): 387. http://dx.doi.org/10.3390/mi10060387.

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Intracellular gene delivery is normally required to study gene functions. A versatile platform able to perform both chemical transfection and viral transduction to achieve efficient gene modification in most cell types is needed. Here we demonstrated that high throughput chemical transfection, virus packaging, and transduction can be conducted efficiently on our previously developed superhydrophobic microwell array chip (SMAR-chip). A total of 169 chemical transfections were successfully performed on the chip in physically separated microwells through a few simple steps, contributing to the convenience of DNA delivery and media change on the SMAR-chip. Efficiencies comparable to the traditional transfection in multi-well plates (~65%) were achieved while the manual operations were largely reduced. Two transfection procedures, the dry method amenable for the long term storage of the transfection material and the wet method for higher efficiencies were developed. Multiple transfections in a scheduled manner were performed to further increase the transfection efficiencies or deliver multiple genes at different time points. In addition, high throughput virus packaging integrated with target cell transduction were also proved which resulted in a transgene expression efficiency of >70% in NIH 3T3 cells. In summary, the SMAR-chip based high throughput gene delivery is efficient and versatile, which can be used for large scale genetic modifications in a variety of cell types.

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4

Gam, Jeremy J., Breanna DiAndreth, Ross D. Jones, Jin Huh i Ron Weiss. "A ‘poly-transfection’ method for rapid, one-pot characterization and optimization of genetic systems". Nucleic Acids Research 47, nr 18 (2.08.2019): e106-e106. http://dx.doi.org/10.1093/nar/gkz623.

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Abstract Biological research is relying on increasingly complex genetic systems and circuits to perform sophisticated operations in living cells. Performing these operations often requires simultaneous delivery of many genes, and optimizing the stoichiometry of these genes can yield drastic improvements in performance. However, sufficiently sampling the large design space of gene expression stoichiometries in mammalian cells using current methods is cumbersome, complex, or expensive. We present a ‘poly-transfection’ method as a simple yet high-throughput alternative that enables comprehensive evaluation of genetic systems in a single, readily-prepared transfection sample. Each cell in a poly-transfection represents an independent measurement at a distinct gene expression stoichiometry, fully leveraging the single-cell nature of transfection experiments. We first benchmark poly-transfection against co-transfection, showing that titration curves for commonly-used regulators agree between the two methods. We then use poly-transfections to efficiently generate new insights, for example in CRISPRa and synthetic miRNA systems. Finally, we use poly-transfection to rapidly engineer a difficult-to-optimize miRNA-based cell classifier for discriminating cancerous cells. One-pot evaluation enabled by poly-transfection accelerates and simplifies the design of genetic systems, providing a new high-information strategy for interrogating biology.

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5

Gardiner, Donald L., Tina S. Skinner-Adams, Tobias Spielmann i Katharine R. Trenholme. "Malaria transfection and transfection vectors". Trends in Parasitology 19, nr 9 (wrzesień 2003): 381–83. http://dx.doi.org/10.1016/s1471-4922(03)00187-9.

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6

Peng, Zhiqiang, Jianping Xiong, Hanzhi Dong i Wuping Li. "Preparation of Polymer Nanocarrier Material and Its Application in B-Cell Lymphoma". Science of Advanced Materials 12, nr 10 (1.10.2020): 1524–34. http://dx.doi.org/10.1166/sam.2020.3877.

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The β-cyclodextrin (β-CD) is coupled with polyethyleneimine (PEI 600 Da) to produce a polymer nanocarrier material (PyD-W) with good biocompatibility and high transfection rate. First, the test was performed to study the influence of different factors on the transfection efficiency of PyD-W materials in terms of cell type and transfection system. Then the effect of adding wheat germ agglutinin on the material-cell membrane binding when transfecting cells by PyD-W materials was studied. The influence of temperature and cell phagocytosis inhibitors on the entire transfection process were taken into consideration at the same time. In the end, the escape ability of PyD-W material carrying plasmid DNA in lysosomes was studied. After analyzing the performance of PyD-W material transfected cells, the appropriate transfection staining conditions was selected to be applied in the study of the inhibitory properties of mouse B-cell lymphoma cells. In the experiment, the results showed that the cell type had a great influence on the material. The N/P ratio should not be set too high. Prolong the transfecting and fostering time appropriately could increase the transfection efficiency. The addition of wheat germ lectin would significantly reduce the enrichment ability on the cell surface of plasmid DNA carried by PyD-W. Increasing the temperature could also increase the transfection efficiency of PyD-W materials, cell phagocytosis inhibitors would not have a significant impact on transfection, and the accumulation of PyD-W materials in lysosomes would be gradually released from lysosomes with time going by. According to the above results, PyD-W carrying plasmid DNA was transfected into mouse B lymphoma cells and normal B cells using similar transfection methods. The results show that B lymphoma cells (38B9) corresponded significant mRNA levels is lower than the mRNA level of normal B cells (P < 0.05). It is detected by cell count and CCK-8 kit that the growth of cells in the group overexpressing PyD-W is significantly inhibited (P < 0.01).

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7

Yin, Jingwen, i Henry R. Henney Jr. "Stable transfection ofAcanthamoeba". Canadian Journal of Microbiology 43, nr 3 (1.03.1997): 239–44. http://dx.doi.org/10.1139/m97-033.

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The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate – DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 μg/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU – Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.Key words: Acanthamoeba, transfection, ubiquitin, promoter, vector, CAT assay.

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8

Nie, Leng, Li Zeng Gao, Xi Yun Yan i Tai Hong Wang. "Functionalized Tetrapod-Like ZnO Nanostructrures for DNA Gene Delivery". Solid State Phenomena 121-123 (marzec 2007): 747–50. http://dx.doi.org/10.4028/www.scientific.net/ssp.121-123.747.

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Amino-modified tetrapod-like ZnO nanostructures were tried as novel carriers for mammalian cell transfections. The nanostructures consisted of four needle-shaped tetrahedrally arranged legs connected at the center. After silica coating and amino modification, ZnO nanostructures complexes bound plasmid DNA through electrostatic interactions in aqueous solution. When mixed with cells, DNA-nanostructures attached easily onto cell membranes and entered the cells for gene expressions. Due to high positive charge densities on surfaces and needle-shaped tetrahedral structures, functionalized ZnO used as carriers for cell transfections with both high transfection efficiency and little cytotoxicity. And a possible transfection machamism was proposed in this report.

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9

Chung, Namjin, Louis Locco, Kevin W. Huff, Steven Bartz, Peter S. Linsley, Marc Ferrer i Berta Strulovici. "An Efficient and Fully Automated High-Throughput Transfection Method for Genome-Scale siRNA Screens". Journal of Biomolecular Screening 13, nr 2 (23.01.2008): 142–48. http://dx.doi.org/10.1177/1087057107312032.

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RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen. ( Journal of Biomolecular Screening 2008:142-148)

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10

Chong, Kevin Wai Yin, Alan Yiu-Wah Lee, Evelyn S. C. Koay, Sze Jee Seet i Nam Sang Cheung. "pH dependent high transfection efficiency of mouse neuroblastomas using TransFectin". Journal of Neuroscience Methods 158, nr 1 (listopad 2006): 56–63. http://dx.doi.org/10.1016/j.jneumeth.2006.05.017.

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11

Moradian, Hanieh, Andreas Lendlein i Manfred Gossen. "Strategies for simultaneous and successive delivery of RNA". Journal of Molecular Medicine 98, nr 12 (4.11.2020): 1767–79. http://dx.doi.org/10.1007/s00109-020-01956-1.

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AbstractAdvanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

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12

Cheung, Winston Y., Owen Hovey, Jonathan M. Gobin, Gauri Muradia, Jelica Mehic, Carole Westwood i Jessie R. Lavoie. "Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression". Stem Cells International 2018 (24.12.2018): 1–10. http://dx.doi.org/10.1155/2018/1310904.

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Background. Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity. Methods. In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid. Results. TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24–36% GFP-expressing cells with a viability of 85–96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system. Discussion. We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.

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13

Maddila, Santhosh Chandar, Chandrashekhar Voshavar, Porkizhi Arjunan, Rashmi Prakash Chowath, Hari Krishna Reddy Rachamalla, Balaji Balakrishnan, Poonkuzhali Balasubramanian, Rajkumar Banerjee i Srujan Marepally. "Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells". Molecules 26, nr 15 (30.07.2021): 4626. http://dx.doi.org/10.3390/molecules26154626.

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Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2–3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

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14

Li, Chen, Biao Qian, Zhao Ni, Qinzhang Wang, Zixiong Wang, Luping Ma, Zhili Liu, Qiang Li i Xinmin Wang. "Construction of recombinant lentiviral vector containing human stem cell leukemia gene and its expression in interstitial cells of cajal". Open Life Sciences 15, nr 1 (25.03.2020): 83–91. http://dx.doi.org/10.1515/biol-2020-0010.

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AbstractThis study aims to construct recombinant lentiviral vectors containing the human stem cell leukemia (SCL) gene and investigate their in vitro transfection efficiency in Interstitial Cells of Cajal (ICC) of guinea pig bladders. In this study, the human SCL gene was successfully cloned, and the recombinant lentivirus GV287-SCL was successfully constructed. The titer of the recombinant lentivirus was 5 × 108 TU /mL. After transfecting the ICCs with the lentiviral vector at different MOIs, the optimal MOI was determined to be 10.0, and the optimal transfection time was determined to be 3 days. The amplification product of the lentivirus transfection group was consistent with the target fragment, indicating that the SCL gene had been successfully introduced into ICCs. In conclusion, the recombinant lentiviral vector GV287-SCL was successfully constructed and transfected into the in vitro cultured ICCs. The successful expression of SCL in ICCs may provide an experimental basis for the in vivo transfection of the SCL gene.

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15

Nybo, Kristie. "Retroviral Transfection". BioTechniques 53, nr 2 (sierpień 2012): 79–80. http://dx.doi.org/10.2144/000113901.

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Kumar, Priti, Arvindhan Nagarajan i Pradeep D. Uchil. "Optical Transfection". Cold Spring Harbor Protocols 2018, nr 12 (grudzień 2018): pdb.top096222. http://dx.doi.org/10.1101/pdb.top096222.

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Dokka, Sujatha, David Toledo, Liying Wang, Xianglin Shi, Chuanshu Huang, Stephen Leonard i Yon Rojanasakul. "Free radical-mediated transgene inactivation of macrophages by endotoxin". American Journal of Physiology-Lung Cellular and Molecular Physiology 279, nr 5 (1.11.2000): L878—L883. http://dx.doi.org/10.1152/ajplung.2000.279.5.l878.

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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.

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Shen, Wen-Juan, Duo-Mei Tian, Le Fu, Biao Jin, Yu Liu, Yun-Sheng Xu, Yong-Bin Ye i in. "Elastin-Derived VGVAPG Fragment Decorated Cell-Penetrating Peptide with Improved Gene Delivery Efficacy". Pharmaceutics 15, nr 2 (16.02.2023): 670. http://dx.doi.org/10.3390/pharmaceutics15020670.

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Cell-penetrating peptides (CPPs) are attractive non-viral gene delivery vectors due to their high transfection capacity and safety. Previously, we have shown that cell-penetrating peptide RALA can be a promising gene delivery vector for chronic wound regeneration application. In this study, we engineered a novel peptide called RALA-E by introducing elastin-derived VGVAPG fragment into RALA, in order to target the elastin-binding protein on the cell surface and thus improve delivery efficacy of RALA. The transfection efficiency of RALA-E was evaluated by transfecting the HEK-293T and HeLa cell lines cells with RALA-E/pDNA complexes and the flow-cytometry results showed that RALA-E significantly increased the transfection efficiency by nearly 20% in both cell lines compared to RALA. Inhibition of pDNA transfection on HEK-293T cells via chlorpromazine, genistein and mβCD showed that the inhibition extent in transfection efficiency was much less for RALA-E group compared to RALA group. In addition, RALA-E/miR-146a complexes showed up to 90% uptake efficiency in macrophages, and can escape from the endosome and enter the nucleus to inhibit the expression of inflammation genes. Therefore, the developed RALA-E peptide has high potential as a safe and efficient vector for gene therapy application.

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Heng, Zealyn Shi-Lin, Joshua Yi Yeo, Darius Wen-Shuo Koh, Samuel Ken-En Gan i Wei-Li Ling. "Augmenting recombinant antibody production in HEK293E cells: optimizing transfection and culture parameters". Antibody Therapeutics 5, nr 1 (1.01.2022): 30–41. http://dx.doi.org/10.1093/abt/tbac003.

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Abstract Background Optimizing recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialization, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterizations and investigations. Methods Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analyzed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day, and common nutrient supplements. Results Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear polyethylenimine in the most optimized parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg, respectively, in a cost-effective manner. With the addition of peptone, κ light chain increased by ~4-fold to 1032 μg, whereas whole antibody increased to a lesser extent by ~2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production. Conclusions Our optimized findings show promise for a more efficient and convenient antibody production method through transfection and culture optimizations that can be incorporated to scale-up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E.

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Bodbin, Sara E., Chris Denning i Diogo Mosqueira. "Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent". Methods and Protocols 3, nr 3 (9.08.2020): 57. http://dx.doi.org/10.3390/mps3030057.

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Twenty years since their first derivation, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown promise in disease modelling research, while their potential for cardiac repair is being investigated. However, low transfection efficiency is a barrier to wider realisation of the potential this model system has to offer. We endeavoured to produce a protocol for improved transfection of hPSC-CMs using the ViafectTM reagent by Promega. Through optimisation of four essential parameters: (i) serum supplementation, (ii) time between replating and transfection, (iii) reagent to DNA ratio and (iv) cell density, we were able to successfully transfect hPSC-CMs to ~95% efficiencies. Transfected hPSC-CMs retained high purity and structural integrity despite a mild reduction in viability, and preserved compatibility with phenotyping assays of hypertrophy. This protocol greatly adds value to the field by overcoming limited transfection efficiencies of hPSC-CMs in a simple and quick approach that ensures sustained expression of transfected genes for at least 14 days, opening new opportunities in mechanistic discovery for cardiac-related diseases.

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Sanderson, Theo, i Julian C. Rayner. "PhenoPlasm: a database of disruption phenotypes for malaria parasite genes". Wellcome Open Research 2 (21.06.2017): 45. http://dx.doi.org/10.12688/wellcomeopenres.11896.1.

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Two decades after the first Plasmodium transfection, attempts have been made to disrupt more than 3,151 genes in malaria parasites, across five Plasmodium species. While results from rodent malaria transfections have been curated and systematised, empowering large-scale analysis, phenotypic data from human malaria parasite transfections currently exists as individual reports scattered across a the literature. To facilitate systematic analysis of published experimental genetic data across Plasmodium species, we have built PhenoPlasm (http://www.phenoplasm.org), a database of phenotypes generated by transfection experiments in all Plasmodium parasites. The site provides a simple interface linking citation-backed Plasmodium reverse-genetic phenotypes to gene IDs. The database has been populated with phenotypic data on 367 P. falciparum genes, curated from 176 individual publications, as well as existing data on rodent Plasmodium species from RMgmDB and PlasmoGEM. This is the first time that all available data on P. falciparum transfection experiments has been brought together in a single place. These data are presented using ortholog mapping to allow a researcher interested in a gene in one species to see results across other Plasmodium species. The collaborative nature of the database enables any researcher to add new phenotypes as they are discovered. As an example of database utility, we use the currently available datasets to identify RAP (RNA-binding domain abundant in Apicomplexa)-domain containing proteins as crucial to parasite survival.

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Sanderson, Theo, i Julian C. Rayner. "PhenoPlasm: a database of disruption phenotypes for malaria parasite genes". Wellcome Open Research 2 (24.07.2017): 45. http://dx.doi.org/10.12688/wellcomeopenres.11896.2.

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Two decades after the first Plasmodium transfection, attempts have been made to disrupt more than 3,151 genes in malaria parasites, across five Plasmodium species. While results from rodent malaria transfections have been curated and systematised, empowering large-scale analysis, phenotypic data from human malaria parasite transfections currently exists as individual reports scattered across a the literature. To facilitate systematic analysis of published experimental genetic data across Plasmodium species, we have built PhenoPlasm (http://www.phenoplasm.org), a database of phenotypes generated by transfection experiments in all Plasmodium parasites. The site provides a simple interface linking citation-backed Plasmodium reverse-genetic phenotypes to gene IDs. The database has been populated with phenotypic data on 367 P. falciparum genes, curated from 176 individual publications, as well as existing data on rodent Plasmodium species from RMgmDB and PlasmoGEM. This is the first time that all available data on P. falciparum transfection experiments has been brought together in a single place. These data are presented using ortholog mapping to allow a researcher interested in a gene in one species to see results across other Plasmodium species. The collaborative nature of the database enables any researcher to add new phenotypes as they are discovered. As an example of database utility, we use the currently available datasets to identify RAP (RNA-binding domain abundant in Apicomplexa)-domain containing proteins as crucial to parasite survival.

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Zhang, Guohong, i Steven R. Kain. "Transfection Maximizer Increases the Efficiency of Calcium Phosphate Transfections with Mammalian Cells". BioTechniques 21, nr 5 (listopad 1996): 940–45. http://dx.doi.org/10.2144/96215pf02.

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Liu, Dexi, Tan Ren i Xiang Gao. "Cationic Transfection Lipids". Current Medicinal Chemistry 10, nr 14 (1.07.2003): 1307–15. http://dx.doi.org/10.2174/0929867033457386.

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Dean, David A., i Joshua Z. Gasiorowski. "Liposome-Mediated Transfection". Cold Spring Harbor Protocols 2011, nr 3 (marzec 2011): prot5583. http://dx.doi.org/10.1101/pdb.prot5583.

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Dean, David A., i Joshua Z. Gasiorowski. "Dendrimer-Mediated Transfection". Cold Spring Harbor Protocols 2011, nr 3 (marzec 2011): prot5584. http://dx.doi.org/10.1101/pdb.prot5584.

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Toma, Tudor. "Transfection using lasers". Genome Biology 3 (2002): spotlight—20020725–01. http://dx.doi.org/10.1186/gb-spotlight-20020725-01.

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Wesson, Dawn M., i Donald J. Krogstad. "Transfection and malaria". Nature Medicine 1, nr 8 (sierpień 1995): 745–47. http://dx.doi.org/10.1038/nm0895-745.

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Won, Rachel. "Single-cell transfection". Nature Photonics 7, nr 10 (27.09.2013): 762. http://dx.doi.org/10.1038/nphoton.2013.260.

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Kumar, Priti, Arvindhan Nagarajan i Pradeep D. Uchil. "DEAE-Dextran Transfection". Cold Spring Harbor Protocols 2018, nr 7 (lipiec 2018): pdb.top096263. http://dx.doi.org/10.1101/pdb.top096263.

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Moguel, Bárbara, Raúl J. Bobes, Julio C. Carrero i Juan P. Laclette. "Transfection of Platyhelminthes". BioMed Research International 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/206161.

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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.

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32

Roberts, Josh P. "Tackling Transfection Tactically". Genetic Engineering & Biotechnology News 32, nr 5 (marzec 2012): 36–37. http://dx.doi.org/10.1089/gen.32.5.15.

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McKenna, Neil. "Tackling Transfection Tasks". Genetic Engineering & Biotechnology News 33, nr 1 (styczeń 2013): 1, 22, 24. http://dx.doi.org/10.1089/gen.33.01.13.

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Labant, Mary Ann. "Transfection Methods Evolving". Genetic Engineering & Biotechnology News 33, nr 15 (wrzesień 2013): 1, 32, 33, 34,. http://dx.doi.org/10.1089/gen.33.15.11.

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35

Roulland-Dussoix, Daisy. "Transfection ofCorynebacterium diphtheriae". Current Microbiology 18, nr 5 (maj 1989): 319–21. http://dx.doi.org/10.1007/bf01575948.

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Rose, John K. "Optimization of Transfection". Current Protocols in Neuroscience 1, nr 1 (listopad 1997): A.1B.1—A.1B.3. http://dx.doi.org/10.1002/0471142301.nsa01bs01.

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Kingston, Robert E., Claudia A. Chen i Hiroto Okayama. "Calcium Phosphate Transfection". Current Protocols in Neuroscience 1, nr 1 (listopad 1997): A.1C.1—A.1C.8. http://dx.doi.org/10.1002/0471142301.nsa01cs01.

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Potter, Huntington. "Transfection by Electroporation". Current Protocols in Neuroscience 1, nr 1 (listopad 1997): A.1E.1—A.1E.5. http://dx.doi.org/10.1002/0471142301.nsa01es01.

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Tinsley, John H., James Hawker i Yuan Yuan. "Efficient protein transfection of cultured coronary venular endothelial cells". American Journal of Physiology-Heart and Circulatory Physiology 275, nr 5 (1.11.1998): H1873—H1878. http://dx.doi.org/10.1152/ajpheart.1998.275.5.h1873.

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Although it is well recognized that microvascular endothelial cells play an important role in the local regulation of tissue perfusion and exchange processes, the precise effect of specific endothelial proteins on microvascular function remains to be elucidated. The lack of information is partially due to methodological limitations, because pharmacological approaches that are routinely used in conventional microcirculatory studies produce nonspecific information. The purpose of this study was to develop an efficient method of transfecting endothelial cells with proteins for functional analysis. TransIT, a polyamine reagent, proved very successful for β-galactosidase (β-Gal) protein transfection of bovine coronary venular endothelial cells, because time-course and dose-dependent experiments showed that a transfection efficiency of 88 ± 7% was possible. In control studies, β-Gal was detected in transfected cells that were trypsinized and washed, indicating that the protein was not merely adhering to the cell surface. Furthermore, transfection of a cell-impermeable peptide inhibitor of protein kinase C (PKC) resulted in a decrease in PKC activity in comparison with control cells. This approach provides a technical basis for further transfection of endothelial cell monolayers with antibodies and constitutively active or dominant-negative proteins to study the molecular control of microvascular function.

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40

Pitt, William G., Daniel R. Jack, Ghaleb A. Husseini i Brett V. Memmott. "Non-Viral Gene Transfection with Ultrasound: Is 100% Transfection Possible?" Advanced Science Letters 11, nr 1 (30.05.2012): 98–105. http://dx.doi.org/10.1166/asl.2012.2161.

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Nimesh, Surendra, Amit Saxena, Ajeet Kumar i Ramesh Chandra. "Improved transfection efficiency of chitosan-DNA complexes employing reverse transfection". Journal of Applied Polymer Science 124, nr 3 (21.10.2011): 1771–77. http://dx.doi.org/10.1002/app.35179.

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Yano, Motoki, Yoshiaki Nakashima, Yoshihiro Kobayashi, Kotaro Mizuno, Akimitsu Konishi, Hidefumi Sasaki, Ichiro Fukai, Ronald K. Scheule i Yoshitaka Fujii. "Endostatin gene transfection using a cationic lipid: advantages of transfection before tumor cell inoculation and repeated transfection". Cancer Gene Therapy 11, nr 5 (26.03.2004): 354–62. http://dx.doi.org/10.1038/sj.cgt.7700704.

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Kościańska, E., i K. Wypijewski. "Electroporated intact BY-2 tobacco culture cells as a model of transient expression study." Acta Biochimica Polonica 48, nr 3 (30.09.2001): 657–61. http://dx.doi.org/10.18388/abp.2001_3900.

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Transfer of foreign genes into plant cells can be accomplished by several methods: agrobacterium-mediated, microinjection, biolistic particle bombardment and electroporation. The last one is frequently used for transfection of plant protoplasts for transient gene expression. Electroporation is a simple procedure and allows transfecting a large number of cells at one time. Square wave-modulated porators are the most efficient for introducing expression cassettes into plant protoplasts. Based on a protocol developed by Wu & Feng (Plant Cell Reports, 1999, 18, 381-386), we optimized conditions for transfection of intact Nicotiana tabacum BY-2 cells using square wave-modulated electroporator. To simplify screening for transfected gene expression we used constructs with a GFP marker gene.

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44

da Silva, Zigomar, Andressa Pereira de Souza, José Rodrigo Claudio Pandolfi, Francisco Noé da Fonseca, Carlos André da Veiga Lima-Rosa i Mariana Groke Marques. "Comparison between electroporation and polyfection in pig sperm: efficiency and cell viability implications". Zygote 26, nr 4 (sierpień 2018): 286–93. http://dx.doi.org/10.1017/s0967199418000205.

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SummaryThe aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 μs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.

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45

Sturm, Lisa, Bettina Schwemberger, Ursula Menzel, Sonja Häckel, Christoph E. Albers, Christian Plank, Jaap Rip i in. "In Vitro Evaluation of a Nanoparticle-Based mRNA Delivery System for Cells in the Joint". Biomedicines 9, nr 7 (8.07.2021): 794. http://dx.doi.org/10.3390/biomedicines9070794.

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Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

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46

Tsan, M. F., J. E. White i B. Shepard. "Lung-specific direct in vivo gene transfer with recombinant plasmid DNA". American Journal of Physiology-Lung Cellular and Molecular Physiology 268, nr 6 (1.06.1995): L1052—L1056. http://dx.doi.org/10.1152/ajplung.1995.268.6.l1052.

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A number of gene delivery methods have been developed to facilitate gene transfer into mammalian somatic cells in vivo. In this study, we demonstrated that tracheal insufflation of two recombinant plasmids containing a bacterial gene, chloramphenicol acetyltransferase (CAT), driven by the cytomegalovirus (CMV) immediate early promoter/enhancer, either alone or complexed to cationic liposomes, resulted in efficient and selective transfection of the lungs in rats. When the simian virus 40 (SV40) promoter/enhancer was used, there was no detectable transfection. Insufflation of plasmid DNA was as efficient as plasmid-liposome complexes in transfecting the lungs. Expression of the CAT gene in the lungs was noted as early as 1 day after insufflation of plasmid DNA alone or plasmid-liposome complexes, and lasted for > 21 days. In contrast, intravenous injection of plasmid alone or plasmid-liposome complexes did not result in transfection of the lungs. Because of its simplicity, without the potential adverse effect of any gene delivery systems, intratracheal delivery of recombinant plasmid DNA may have potential implication for lung-specific gene therapy.

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47

Van der Loo, Johannes C. M., William Swaney, Diana Nordling, Axel Schambach, Christopher Baum, David A. Williams, Lilith Reeves i Punam Malik. "Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor". Blood 112, nr 11 (16.11.2008): 3539. http://dx.doi.org/10.1182/blood.v112.11.3539.3539.

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Abstract The need for gamma-retroviral vectors with self-inactivating (SIN) long terminal repeats for clinical trials has prompted a shift in the method with which large scale GMP-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN retrovirus vectors that produced high amounts of genomic vector RNA in packaging cells, and to design scalable processes for closed system culture, transfection and virus harvest. Using improved expression plasmids, the Vector Production Facility, an academic GMP manufacturing laboratory that is part of the Translational Cores at Cincinnati Children’s Hospital, has developed such a method based on the Wave Bioreactor® production platform. In brief, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave Cell Bag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16–20 hrs post-transfection, the media is changed; virus is harvested at 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted into Cryocyte freezing containers, and frozen at or below −70°C. Several processing parameters, including the confluency of cells harvested prior to transfection, the timing of transfection, the amount of plasmid DNA, exposure of cells to PBS/TrypLESelect, and the timing of the media change post-transfection affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding (as is standard when using tissue culture plastic) increased the titer from 104 to 4 × 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 μg doubled the titer in the Wave, while it reduced titer by 20–40% in tissue culture flasks (Fig. 1). Using an optimized protocol, six cGMP-grade SIN gamma-retroviral vectors have now been produced in support of the FDA’s National Toxicology Program (NTP), with unconcentrated vector titers ranging from 1 × 106 to as high as 4 × 107 IU/mL. Using similar processing, we have produced a large scale SIN gamma-retroviral vector (GALV pseudotyped) for an international X-linked SCID trial with average unconcentrated titers of 106 IU/mL in all viral harvests. In summary, the process developed at the Cincinnati Children’s Hospital Vector Production Facility allows for large scale closed-system production of high-titer retroviral vectors for clinical trials using transient transfection. Figure Figure

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Sork, Helena, Joel Z. Nordin, Janne J. Turunen, Oscar PB Wiklander, Burcu Bestas, Eman M. Zaghloul, Helerin Margus i in. "Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency". Molecular Therapy - Nucleic Acids 5 (2016): e290. http://dx.doi.org/10.1038/mtna.2016.8.

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Bruggencate, Filip ten, Fabrice Laroche, Yue Zhang, Guoqiang Song, Shougen Yin, Jan Pieter Abrahams i Zunfeng Liu. "Visualizing the localization of transfection complexes during graphene nanoparticle-based transfection". Journal of Materials Chemistry B 1, nr 46 (2013): 6353. http://dx.doi.org/10.1039/c3tb21349h.

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McNaughton, B. R., J. J. Cronican, D. B. Thompson i D. R. Liu. "Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins". Proceedings of the National Academy of Sciences 106, nr 15 (23.03.2009): 6111–16. http://dx.doi.org/10.1073/pnas.0807883106.

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