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Fluorescent labeling of DNA : strategies, pitfalls and necessity for fluorescence microscopy investigations of gene therapy

Koen Rombouts (UGent)
(2016)
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(UGent) and (UGent)
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Abstract
Gene therapy is a field of research in which a huge amount of effort has been spent over the last two decades. To succeed, a correct therapeutic gene needs to be delivered into the cytoplasm of a cell for mRNA and nucleus of a cell for plasmid DNA (pDNA). To protect the nucleic acids, viral vectors and non-viral carriers have been used. Due to the many barriers that are encountered in the delivery, the design and evaluation of especially the non-viral vectors has room for improvement. To this end, fluorescence microscopy has proven to be a useful tool. In this thesis, the main goal was to study the degradation of pDNA with advanced microscopy. Since fluorescence microscopy requires a fluorescent tag on the molecules of interest, an overview of the possible nucleic acid labeling strategies was given. Attention was given to the effect the methods have on the intracellular processing. This is studied in detail for a frequently used random covalent labeling method. When using Lipofectamine, the transfection efficiency drops for high labeling densities, probably due to an increased hydrophobicity which causes a higher affinity for lipid structures and steric hindrance of the labels for transcriptional proteins. Alternative pDNA labeling strategies were also explored. Fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) were evaluated for their ability to follow pDNA degradation. SPT could measure the degradation of pDNA in cell lysate after lipofection. Finally, the gene delivery potential of 3 polycationic amphiphilic β-cyclodextrins (CDs) was tested for pDNA and mRNA. Due to a lower cellular uptake, no transfection could be induced in the presence of serum. It was seen that the interactions of CDs with cellular cholesterol are likely blocked by the serum proteins. In conclusion, we have shown that the correct use of microscopy methods, and especially SPT, is valuable in the study and evaluation of barriers for non-viral nucleic acid carriers.
Keywords
Fluorescence microscopy methods, DNA labeling, Gene therapy

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Citation

Please use this url to cite or link to this publication:

Chicago
Rombouts, Koen. 2016. “Fluorescent Labeling of DNA : Strategies, Pitfalls and Necessity for Fluorescence Microscopy Investigations of Gene Therapy”. Ghent, Belgium: Ghent University. Faculty of Pharmaceutical Sciences.
APA
Rombouts, K. (2016). Fluorescent labeling of DNA : strategies, pitfalls and necessity for fluorescence microscopy investigations of gene therapy. Ghent University. Faculty of Pharmaceutical Sciences, Ghent, Belgium.
Vancouver
1.
Rombouts K. Fluorescent labeling of DNA : strategies, pitfalls and necessity for fluorescence microscopy investigations of gene therapy. [Ghent, Belgium]: Ghent University. Faculty of Pharmaceutical Sciences; 2016.
MLA
Rombouts, Koen. “Fluorescent Labeling of DNA : Strategies, Pitfalls and Necessity for Fluorescence Microscopy Investigations of Gene Therapy.” 2016 : n. pag. Print.
@phdthesis{7246373,
  abstract     = {Gene therapy is a field of research in which a huge amount of effort has been spent over the last two decades. To succeed, a correct therapeutic gene needs to be delivered into the cytoplasm of a cell for mRNA and nucleus of a cell for plasmid DNA (pDNA). To protect the nucleic acids, viral vectors and non-viral carriers have been used. Due to the many barriers that are encountered in the delivery, the design and evaluation of especially the non-viral vectors has room for improvement. To this end, fluorescence microscopy has proven to be a useful tool. In this thesis, the main goal was to study the degradation of pDNA with advanced microscopy. Since fluorescence microscopy requires a fluorescent tag on the molecules of interest, an overview of the possible nucleic acid labeling strategies was given. Attention was given to the effect the methods have on the intracellular processing. This is studied in detail for a frequently used random covalent labeling method. When using Lipofectamine, the transfection efficiency drops for high labeling densities, probably due to an increased hydrophobicity which causes a higher affinity for lipid structures and steric hindrance of the labels for transcriptional proteins. Alternative pDNA labeling strategies were also explored. Fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) were evaluated for their ability to follow pDNA degradation. SPT could measure the degradation of pDNA in cell lysate after lipofection. Finally, the gene delivery potential of 3 polycationic amphiphilic β-cyclodextrins (CDs) was tested for pDNA and mRNA. Due to a lower cellular uptake, no transfection could be induced in the presence of serum. It was seen that the interactions of CDs with cellular cholesterol are likely blocked by the serum proteins.
In conclusion, we have shown that the correct use of microscopy methods, and especially SPT, is valuable in the study and evaluation of barriers for non-viral nucleic acid carriers.},
  author       = {Rombouts, Koen},
  keywords     = {Fluorescence microscopy methods,DNA labeling,Gene therapy},
  language     = {eng},
  pages        = {253},
  publisher    = {Ghent University. Faculty of Pharmaceutical Sciences},
  school       = {Ghent University},
  title        = {Fluorescent labeling of DNA : strategies, pitfalls and necessity for fluorescence microscopy investigations of gene therapy},
  year         = {2016},
}