Intracellular distribution of DNA internalized through calcium phosphate precipitation.
Although calcium phosphate precipitation is the most commonly used method for DNA-mediated gene transfer, the mechanism for its action is unknown. We showed recently that both transient and stable expression of exogenous genes in the transfected cells are entirely dependent on DNA internalized through active endocytosis. We now report on the subcellular distribution of the endocytosed DNA. After exposure to calcium phosphate-precipitated DNA, cultured fibroblasts internalized less than 10% of the DNA into the nuclei fraction. About 20% was recovered in each of the putative plasma membrane and vesicular organelle fractions. Although over 50% was recovered in the cytosolic fraction, it was completely degraded to oligonucleotides of smaller than 100 bp. In contrast, intact DNA molecules were recovered in all the other subcellular fractions. Similar patterns of DNA distribution were observed not only in the easily transformed mouse cells (Ltk-) but also in the transformation-resistant human primary fibroblasts. In conclusion, DNA-mediated gene transfer by calcium phosphate precipitation is an inefficient procedure because over 50% of the DNA is almost immediately degraded and released into the cytosol. Contrary to accepted views, DNA macromolecules do not seem to pass through the cytosol before reaching the nuclei. A novel transport pathway is proposed in which exogenous DNA molecules may be transferred directly by intermediary vesicles from the endocytic-lysosomal compartment to the nucleus.
Orrantia
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Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada.
Chang
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D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015139:Blotting, Southern; D002130:Calcium Phosphates / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D011232:Chemical Precipitation; D004247:DNA / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D051379:Mice; D010957:Plasmids
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D004247:DNA; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous