Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.
Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.
Cohen
Richard N
RN
Joint Graduate Group in Bioengineering, University of California at San Francisco, San Francisco, CA 94143-0912, USA.
van der Aa
Marieke A E M
MA
Macaraeg
Nichole
N
Lee
Ai Ping
AP
Szoka
Francis C
FC
D000818:Animals; D001693:Biological Transport, Active; D002277:Carcinoma / Q000473:pathology; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D003287:Contrast Media / Q000737:chemistry; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D003902:Detergents / Q000737:chemistry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008175:Lung Neoplasms / Q000473:pathology; D008546:Melanoma, Experimental / Q000473:pathology; D051379:Mice; D008970:Molecular Weight; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000592:standards; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D014283:Triiodobenzoic Acids / Q000737:chemistry
D003287:Contrast Media; D003902:Detergents; D014283:Triiodobenzoic Acids; D011094:Polyethyleneimine; D004247:DNA; D008156:Luciferases; C044834:iodixanol