Prolonged transgene expression in rodent lung cells.
We tested the efficiency of several different cationic liposome formulations, complexed to one of two different chloramphenicol acetyltransferase (CAT) reporter plasmids, in transfecting freshly isolated, highly purified rat lung alveolar type II cells, alveolar macrophages, and three different human lung carcinoma cell lines, as well as NIH 3T3 cells, a rapidly dividing, transformed mouse fibroblast line. Our results demonstrated that several different cationic liposome formulations can mediate high-level CAT gene expression in all the cell types tested. Electron microscopic analysis confirmed that cationic liposome-DNA complexes are avidly bound and internalized by lung cells. The time course of expression of transfected genes in nontransformed cell types with low mitotic indices, such as type II cells, is poorly characterized. NIH 3T3 cells expressed maximal CAT activity by day 4 following transfection, with virtual disappearance of activity by day 11. Conversely, type II cells expressed maximal CAT activity between days 5 and 11, and CAT activity was still clearly present 35 days after transfection. Southern blot analysis of DNA isolated from transfected type II cells revealed that the CAT gene was largely present in an extrachromosomal form, rather than integrated into genomic DNA. These observations indicate that following cationic liposome-mediated transfection, rat alveolar type II cells (the majority of which do not divide in culture) can express transfected genes for prolonged periods, apparently mediated by expression of the transgene in an episomal form.
Debs
R
R
Howard Hughes Medical Institute, Department of Pediatrics, University of California, San Francisco 94143-0128.
Pian
M
M
Gaensler
K
K
Clements
J
J
Friend
D S
DS
Dobbs
L
L
D016475:3T3 Cells; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D015139:Blotting, Southern; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D008168:Lung / Q000166:cytology / Q000502:physiology*; D008175:Lung Neoplasms; D016676:Macrophages, Alveolar / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D008051:Lipid Bilayers; D008081:Liposomes; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase