title: Live Cell Characterization of DNA Aggregation Delivered through Lipofection.
abstract: DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.
authors.affiliation: University of Western Sydney, School of Science and Health, Hawkesbury Campus, Locked Bag 1797, Penrith NSW 2751, Australia.
authors.forename: Stephen
authors.initials: S
authors.lastname: Mieruszynski
authors.affiliation: University of Western Sydney, School of Science and Health, Hawkesbury Campus, Locked Bag 1797, Penrith NSW 2751, Australia.
authors.forename: Candida
authors.initials: C
authors.lastname: Briggs
authors.affiliation: 1] Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America; Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California, United States of America [2] Centre for Bioactive Discovery in Health and Ageing, School of Science &Technology, University of New England, Armidale, Australia.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.affiliation: 1] Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America; Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California, United States of America [2] Centre for Bioactive Discovery in Health and Ageing, School of Science &Technology, University of New England, Armidale, Australia.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: University of Western Sydney, School of Science and Health, Hawkesbury Campus, Locked Bag 1797, Penrith NSW 2751, Australia.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Jones
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013388:Succinimides / Q000737:chemistry; D014162:Transfection / Q000379:methods*
chemical_list: C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D013388:Succinimides; D004247:DNA