In the development of non-viral gene delivery vectors, it is essential to reliably localize and quantify transfected DNA inside the cell. To track DNA, fluorescence microscopy methods are commonly applied. These mostly rely on fluorescently labeled DNA, DNA binding proteins fused to a fluorescent protein, or fluorescence in situ hybridization (FISH). In addition, co-stainings are often used to determine the colocalization of the DNA in specific cellular compartments, such as the endolysosomes or the nucleus. We provide an overview of these DNA tracking methods, advice on how they should be combined, and indicate which co-stainings or additional methods are required to draw precise conclusions from a DNA tracking experiment. Some emphasis is given to the localization of exogenous DNA inside the nucleus, which is the last step of DNA delivery. We argue that suitable tools which allow for the nuclear detection of faint signals are still missing, hampering the rational development of more efficient non-viral transfection systems.
Keywords: DNA visualization; DNA-binding proteins; Fluorescence in situ hybridization; Fluorescence microscopy; Gene delivery; Labeled DNA; Non-viral vectors.
© 2024. The Author(s).