title: Efficiency of plasmid delivery and expression after lipid-mediated gene transfer to human cells in vitro.
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.
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Bebök
authors.forename: A M
authors.initials: AM
authors.lastname: Abai
authors.forename: J Y
authors.initials: JY
authors.lastname: Dong
authors.forename: S A
authors.initials: SA
authors.lastname: King
authors.forename: K L
authors.initials: KL
authors.lastname: Kirk
authors.forename: G
authors.initials: G
authors.lastname: Berta
authors.forename: B W
authors.initials: BW
authors.lastname: Hughes
authors.forename: A S
authors.initials: AS
authors.lastname: Kraft
authors.forename: S W
authors.initials: SW
authors.lastname: Burgess
authors.forename: W
authors.initials: W
authors.lastname: Shaw
authors.forename: P L
authors.initials: PL
authors.lastname: Felgner
authors.forename: E J
authors.initials: EJ
authors.lastname: Sorscher
mesh_terms: D002460:Cell Line; D004274:DNA, Recombinant / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D015793:HLA-B7 Antigen / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D015793:HLA-B7 Antigen; D008081:Liposomes; D001616:beta-Galactosidase