Intracellular compartmentalization of DNA fragments in cultured airway epithelial cells mediated by cationic lipids.
OBJECTIVE
The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection.


METHODS
Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine at various charge ratios.


RESULTS
A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.
Holmes
A R
AR
Gene Therapy Core Center, Cardiovascular Research Institute, University of California, San Francisco 94143, USA.
Dohrman
A F
AF
Ellison
A R
AR
Goncz
K K
KK
Gruenert
D C
DC
D001980:Bronchi / Q000378:metabolism* / Q000473:pathology; D002411:Cation Exchange Resins / Q000008:administration & dosage* / Q000493:pharmacokinetics; D002412:Cations; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000493:pharmacokinetics; D004337:Drug Carriers; D004355:Drug Stability; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008055:Lipids / Q000008:administration & dosage* / Q000493:pharmacokinetics; D009841:Oligonucleotides / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics; D010761:Phosphorus Radioisotopes; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D002411:Cation Exchange Resins; D002412:Cations; D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D004247:DNA