Expression of human growth hormone fusion genes in cultured lung endothelial cells and in the lungs of mice.
We sought to develop genetic therapy for acute lung diseases by introducing genes into lung cells in vivo that were only transiently expressed. To that end, we introduced a gene encoding a physiologically relevant secreted human protein into bovine lung endothelial cells in culture and into the lungs of mice using the technique of lipofection. We exposed cultured endothelial cells to a plasmid containing the coding region for human growth hormone (hGH) driven by a metallothionein (MT) promoter. In cells lipofected with the plasmid containing the MT promoter, expression of the hGH gene in medium was low (peak = 30 ng hGH/24 h/60-mm dish), but expression was markedly increased by addition of either dexamethasone (peak = 91) or cadmium (peak = 120). Lipofection with the same construct except a thymidine kinase promoter showed no cadmium response. We gave mice 5,000 ppm ZnSO4 in their drinking water and 24 h later injected intravenously plasmid containing the MT promoter complexed to liposomes. Mice were killed 1, 3, and 5 days after injection, and hGH production by minced lung, liver, and kidneys was determined in vitro. Neither kidneys nor liver produced detectable hGH. However, hGH was produced by the lungs, beginning on day 1, peaking on day 3 (approximately 1.0 ng hGH/24 h/g tissue), and declining by day 5. Lungs from mice injected either with DNA alone or with liposome alone did not produce hGH. mRNA specific for hGH was demonstrated in the lungs by polymerase chain reaction amplification of cDNA followed by agarose gel electrophoreses.(ABSTRACT TRUNCATED AT 250 WORDS)
Brigham
K L
KL
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2650.
Meyrick
B
B
Christman
B
B
Conary
J T
JT
King
G
G
Berry
L C
LC
Magnuson
M A
MA
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D014162:Transfection
D012313:RNA; D013006:Growth Hormone; D004247:DNA; D008668:Metallothionein