Efficiency of plasmid delivery and expression after lipid-mediated gene transfer to human cells in vitro.

Academic Article

Abstract

  • Cationic liposome-mediated gene transfer has become increasingly important in the development of experimental therapies for human diseases, such as melanoma, human immunodeficiency virus infection, cystic fibrosis and alpha-1 antitrypsin deficiency. However, very little is known about the mechanisms by which lipid-mediated gene transfer occurs. We studied the kinetics of plasmid delivery and expression by using this technique. Plasmid entry in the cystic fibrosis respiratory epithelial cell line 2CFSME0-1 as well as in two other cell lines (HeP 2g and HeLa) occurred in 95 to 100% of cells within 1 hr of the initiation of lipid-mediated gene transfer. In hepatic and respiratory cells, transcription of a construct containing the cystic fibrosis transmembrane conductance regulator was observed in more than 80% of the cell population; similarly high levels of plasmid utilization were obtained in studies of HLA-B7 expression in human melanoma cells. Studies directly relevant to current human trials of lipid-mediated gene transfer indicate that plasmid entry, transcription and translation are often surprisingly efficient, and may occur in nearly 100% of human cells in culture when sensitive methods for detection are used. Furthermore, conventional X-gal immunohistochemistry markedly underestimates transfection efficiency during transient gene expression. These studies point to a new mechanistic understanding of the features that limit expression by using cationic liposomes.
  • Keywords

  • Cell Line, DNA, Recombinant, Gene Transfer Techniques, HLA-B7 Antigen, HeLa Cells, Humans, Liposomes, Plasmids, beta-Galactosidase
  • Author List

  • Bebök Z; Abai AM; Dong JY; King SA; Kirk KL; Berta G; Hughes BW; Kraft AS; Burgess SW; Shaw W
  • Start Page

  • 1462
  • End Page

  • 1469
  • Volume

  • 279
  • Issue

  • 3