Nonviral gene transfer into primary cultures of human and porcine mesothelial cells.
Due to their abundance and accessibility, mesothelial cells may be suitable tools for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter. Transfection was achieved using cationic lipids (DOSPER and DOTAP) or calcium phosphate/deoxyribonucleic acid coprecipitation or Fugene 6. Results showed that Fugene 6 was the most efficient and reproducible transfection reagent with both PMCs and HMCs. With Fugene 6, luciferase activity in PMCs (1.5 x 10(8) relative light units [RLU]/10(6) cells) was at least 2.5-fold higher than with the other transfection reagents, and it was 100-fold higher than in HMCs. However, the proportion of transfected cells expressing GFP was only 1%. These preliminary findings open up new avenues for developing experimental studies on the use of genetically modified PMCs.
Ohan
J
J
Laboratoire de Chirurgie Vasculaire et Thoracique, Paris, France.
Gilbert
M A
MA
Leseche
G
G
Panis
Y
Y
Midoux
P
P
Drouet
L
L
D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds; D013552:Swine; D014162:Transfection
C118665:1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide; D005229:Fatty Acids, Monounsaturated; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D000644:Quaternary Ammonium Compounds; D049452:Green Fluorescent Proteins; D008156:Luciferases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane