Identification of compartments involved in mammalian subcellular trafficking pathways by indirect immunofluorescence

A characteristic of a successful DNA vaccine is its trafficking to the nucleus where it can be transcribed. Plasmid DNA coupled to a delivery vector must enter the cell, navigate its way through endocytic compartments, and ultimately reach the nucleus. Currently, the precise pathway taken by plasmid DNA is not clear. Understanding how plasmid DNA interacts with the cell and which path it follows to reach the nucleus will aid in the rational design of improved delivery vectors. Achieving this goal requires a means by which to monitor the subcellular trafficking of plasmid DNA and delivery vectors. Presented here are methods for identifying various endocytic compartments involved in mammalian subcellular trafficking pathways using indirect immunofluorescence. Together with labeled delivery vectors and/or plasmid DNA, these methods can aid in the understanding of the trafficking pathways involved in DNA delivery, and contribute to the rational design of more efficient delivery vectors.