The efficiency of nuclear plasmid DNA delivery is a critical determinant of transgene expression at the single cell level.
BACKGROUND
The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression.


METHODS
Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery.


RESULTS
PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids.


CONCLUSIONS
Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.
Glover
Dominic J
DJ
Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia.
Leyton
Denisse L
DL
Moseley
Gregory W
GW
Jans
David A
DA
D002467:Cell Nucleus / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004058:Diffusion; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D010957:Plasmids / Q000235:genetics*; D055550:Protein Stability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000235:genetics*
D004247:DNA