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Title: Correlation between cationic lipid-based transfection and cell division

Abstract

We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.

Authors:
; ;
Publication Date:
OSTI Identifier:
22648584
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 345; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BIOMEDICAL RADIOGRAPHY; CELL CYCLE; COMPARATIVE EVALUATIONS; CORRELATIONS; EFFICIENCY; FLUORESCENCE; GENE THERAPY; GENES; IMAGE PROCESSING; IMAGES; LIPIDS; MICROSCOPY; MITOSIS; PROTEINS; STATISTICS

Citation Formats

Kirchenbuechler, Inka, Kirchenbuechler, David, and Elbaum, Michael, E-mail: michael@elbaum.ac.il. Correlation between cationic lipid-based transfection and cell division. United States: N. p., 2016. Web. doi:10.1016/J.YEXCR.2014.11.019.
Kirchenbuechler, Inka, Kirchenbuechler, David, & Elbaum, Michael, E-mail: michael@elbaum.ac.il. Correlation between cationic lipid-based transfection and cell division. United States. doi:10.1016/J.YEXCR.2014.11.019.
Kirchenbuechler, Inka, Kirchenbuechler, David, and Elbaum, Michael, E-mail: michael@elbaum.ac.il. 2016. "Correlation between cationic lipid-based transfection and cell division". United States. doi:10.1016/J.YEXCR.2014.11.019.
@article{osti_22648584,
title = {Correlation between cationic lipid-based transfection and cell division},
author = {Kirchenbuechler, Inka and Kirchenbuechler, David and Elbaum, Michael, E-mail: michael@elbaum.ac.il},
abstractNote = {We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.},
doi = {10.1016/J.YEXCR.2014.11.019},
journal = {Experimental Cell Research},
number = 1,
volume = 345,
place = {United States},
year = 2016,
month = 7
}
  • Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by {approx}10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoylmore » 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L{sub {alpha}} {yields} H{sub II}, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L{sub {alpha}} {yields} H{sub II} phase transition and promoted formation of an inverted cubic phase between the L{sub {alpha}} and H{sub II} phases. In contrast, moderate and weak transfection agents retained the direct L{sub {alpha}} {yields} H{sub II} transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.« less
  • Butylated hydroxytoluene (BHT) is an effective membrane lipid perturber. Uptake studies using (/sup 3/H)BHT showed that it is effectively taken up by V79 Chinese hamster lung cells. The correlation time of rotation (tau/sub c/) of the spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) was decreased 4.0 and 12.9% by addition of 0.03 and 0.07 mM BHT, respectively. This corresponds to increases in membrane fluidity produced by temperature increases of 1.8 and 6.0/sup 0/C, respectively. Neither BHT treatment sensitized the cells to hyperthermia. Also no decrease in membrane lipid fluidity, again as measured with the spin label 2N14, was found in thermotolerant V79 cellsmore » compared to control cells. Thermotolerance was induced by a 20-min exposure to 44.5/sup 0/C and the membrane fluidity measurements were made 16 hour later, when the maximum level of thermotolerance was observed. Thus no evidence was found for a correlation between membrane lipid fluidity and hyperthermic killing of V79 cells.« less
  • No abstract prepared.