通过脂质体转染递送的DNA聚集的活细胞表征

Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

作者信息

Mieruszynski Stephen, Briggs Candida, Digman Michelle A, Gratton Enrico, Jones Mark R

机构信息

University of Western Sydney, School of Science and Health, Hawkesbury Campus, Locked Bag 1797, Penrith NSW 2751, Australia.

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.

出版信息

Sci Rep. 2015 May 27;5:10528. doi: 10.1038/srep10528.

DOI:10.1038/srep10528

PMID:26013547

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4444954/

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.

摘要

DNA转运现象，如DNA聚集发生的位置和程度等信息，在活细胞中尚未得到充分表征。在这里，我们通过应用粒子数与亮度（N&B）方法来表征通过脂质转染递送的DNA的聚集情况。N&B分析表明，在整个活细胞中存在广泛的聚集现象，DNA簇集中在细胞末端和核周区域。一旦进入细胞核，聚集程度降低了3倍。此外，我们发现增加细胞培养基中的血清浓度会导致更大程度的细胞质聚集。此外，还探究了DNA片段大小对聚集的影响，结果显示较大的DNA构建体聚集程度较低。本研究首次对通过脂质转染递送至活细胞中的DNA聚集情况进行了定量分析。此外，本研究还提出了一种该成像方法的其他应用模型，该方法最初是为研究蛋白质寡聚化和聚集而开发的。