The effect of cell division on the cellular dynamics of microinjected DNA and dextran.
Gene delivery is a multistep process that is being studied to increase its efficiency, a major hurdle for effective gene therapy. Our study focused on the nuclear entry step by microinjecting a mixture of fluorescent dextran and the pEYFP-Nuc plasmid (encoding a nuclear-targeted, enhanced GFP) into the cytoplasm of nondividing and dividing cells that were selected using non-chemical means. After 10 and 1000 ng/microl of plasmid DNA (pDNA) were cytoplasmically injected, 28% and 50% of the cells that had not divided expressed GFP, respectively, compared with 50% and 90% for the cells that had divided. This result suggested that pDNA can enter the nonmitotic nuclei of mononucleated cells, albeit at a lower efficiency than mitotic nuclei. The ability of pDNA to enter the intact nuclei of nondividing cells is consistent with our previous experience using multinucleated myotubes and digitonin-permeabilized cells in culture and using intravascular naked pDNA delivery in vivo. An explanation for the small effect of cell division was provided by studies using fluorescently labeled molecules and confocal fluorescent microscopy. They showed that the bulk of large dextran, and similarly pDNA, was excluded from re-formed nuclei after mitosis, thereby limiting the effect of cell division on the nuclear entry of pDNA.
Ludtke
James J
JJ
Departments of Pediatrics and Medical Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Sebestyén
Magdolna G
MG
Wolff
Jon A
JA
D002455:Cell Division / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D003911:Dextrans; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000648:ultrastructure*; D010957:Plasmids / Q000008:administration & dosage / Q000378:metabolism*
D003911:Dextrans; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA