scholarly journals Dielectrophoretic Manipulation of Cell Transfection Efficiency During Electroporation Using a Center Needle Electrode

2021 ◽  
Vol 11 (15) ◽  
pp. 7015
Author(s):  
Eivina Radzevičiūtė ◽  
Arūnas Murauskas ◽  
Paulius Ruzgys ◽  
Saulius Šatkauskas ◽  
Irutė Girkontaitė ◽  
...  
Keyword(s):  
Exposure Time ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Needle Electrode ◽  
Cell Transfection ◽  
Electric Pulses ◽  
Long Duration ◽  
Efficiency Increase

Long duration electric pulses are frequently used to facilitate DNA electrotransfer into cells and tissues, while electroporation pulses can be combined with electrophoresis to maximize the transfection efficiency. In this work, we present the dielectrophoresis (DEP)-assisted methodology for electrotransfer of plasmid DNA (3.5 kbp pmaxGFP) into mammalian cells (CHO-K1). A prototype of an electroporation cuvette with center needle electrode for DEP-assisted transfection is presented resulting in a 1.4-fold of transfection efficiency increase compared to the electroporation-only procedure (1.4 kV/cm × 100 µs × 8). The efficiency of transfection has been compared between three DEP frequencies of 1, 100, and 1 MHz. Lastly, the effects of exposure time (1, 3, and 5 min) during the DEP application step have been determined. It is concluded that the proposed methodology and exposure setup allow a significant improvement of transfection efficiency and could be used as an alternative to the currently popular electrotransfection techniques.

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.

Cationic Gelatin as a Gene Carrier

1995 ◽  
Vol 394 ◽  
Author(s):  
K. E. Brown ◽  
J. Bathon ◽  
C. H. Huang ◽  
R. Dalai ◽  
K. W. Leong
Keyword(s):  
Mammalian Cells ◽  
Viral Vector ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Cell Transfection ◽  
Serum Free Medium ◽  
Free Medium ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Dye Reduction

AbstractCationic gelatin was evaluated as a non-viral vector for cell transfection. We hypothesized that cationic gelatin would be a nontoxic alternative to already existing viral and non-viral cationic vectors. Cationic gelatin was synthesized by modifying gelatin with hexanediamine. Complexation of cationic gelatin with psv-β-gal plasmid caused an electrophoretic mobility shift of the plasmid. Cationic gelatin/DNA complexes were optimized in terms of transfection efficiency in CHODUK XB1 and COS 7 cell lines. Maximal gene expression for both cell types occurred in serum free medium with chloroquine (100 μM) at cationic gelatin/DNA ratios of approximately 2 and 7. In comparison with DEAE dextran, polylysine and Lipofectamine, cationic gelatin was the most efficient in transfecting COS 7 cells, with up to 18% cells transfected. In a dye reduction cytotoxicity assay, cationic gelatin caused < 5% of cells to become nonviable at a concentration of 100 μg/ml, while the other transfection reagents tested at the same concentration caused 25–100% of cell death. These results suggest that cationic gelatin holds promise as an effective vehicle for gene delivery to mammalian cells.

scholarly journals Investigation of Plasmid DNA Delivery and Cell Viability Dynamics for Optimal Cell Electrotransfection In Vitro

2020 ◽  
Vol 10 (17) ◽  
pp. 6070
Author(s):  
Sonam Chopra ◽  
Paulius Ruzgys ◽  
Martynas Maciulevičius ◽  
Milda Jakutavičiūtė ◽  
Saulius Šatkauskas
Keyword(s):  
Cell Membrane ◽  
Cell Viability ◽  
High Voltage ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Dna Delivery ◽  
Output Parameter ◽  
Gene Electrotransfer ◽  
Electric Pulses

Electroporation is an effective method for delivering plasmid DNA molecules into cells. The efficiency of gene electrotransfer depends on several factors. To achieve high transfection efficiency while maintaining cell viability is a tedious task in electroporation. Here, we present a combined study in which the dynamics of both evaluation types of transfection efficiency and the cell viability were evaluated in dependence of plasmid concentration as well as at the different number of high voltage (HV) electric pulses. The results of this study reveal a quantitative sigmoidal (R2 > 0.95) dependence of the transfection efficiency and cell viability on the distance between the cell membrane and the nearest plasmid. We propose this distance value as a new, more accurate output parameter that could be used in further optimization studies as a predictor and a measure of electrotransfection efficiency.

scholarly journals Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

2010 ◽  
Vol 1800 (11) ◽  
pp. 1210-1219 ◽  
Author(s):  
Bennett L. Ibey ◽  
Andrei G. Pakhomov ◽  
Betsy W. Gregory ◽  
Vera A. Khorokhorina ◽  
Caleb C. Roth ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nanosecond Duration ◽  
Selective Cytotoxicity ◽  
Electric Pulses

Hamster polyomavirus-derived virus-like particles are able to transfer in vitro encapsidated plasmid DNA to mammalian cells

Virus Genes ◽  
2007 ◽  
Vol 34 (3) ◽  
pp. 303-314 ◽  
Author(s):  
Tatyana Voronkova ◽  
Andris Kazaks ◽  
Velta Ose ◽  
Muhsin Özel ◽  
Siegfried Scherneck ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Virus Like Particles ◽  
Hamster Polyomavirus

Enhanced nuclear import and transfection efficiency of plasmid DNA using streptavidin-fused importin-β

2005 ◽  
Vol 103 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Takeshi Nagasaki ◽  
Takeshi Kawazu ◽  
Taro Tachibana ◽  
Seizo Tamagaki ◽  
Seiji Shinkai
Keyword(s):  
Plasmid Dna ◽  
Nuclear Import ◽  
Transfection Efficiency
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