Transfection of myoblasts in primary culture with isomeric cationic cholesterol derivatives.
Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three isomers in the case of spermidine and two isomers for spermine that were separated by reversed-phase high-performance liquid chromatography (rp-HPLC). The position of the cholesteryl moiety was assigned by 13C-NMR and coelution with synthetic isomers of defined structure. The isomeric cationic lipids were evaluated for their transfection activity in myoblasts from dog muscle and a human lung epithelial cell line (A549) using plasmid DNA expressing the luciferase reporter gene. The results showed that the position of the cholesteryl moiety is of critical importance for efficient transfection of myoblasts in primary culture with isomers having a derivatized secondary amine being significantly more effective than those with a derivatized primary amine. On the contrary, differences in the A549 cell line were less pronounced and did not follow the same pattern. The results show that slight structural differences between cationic lipids lead to significantly different transfection efficiencies for myoblasts in primary culture. This may also represent an advantage in view of cell or organ targeting.
Bischoff
R
R
Transgene S.A., 11 rue de Molsheim, Cedex, 67082 Strasbourg, France. bischoff@transgene.fr
Cordier
Y
Y
Perraud
F
F
Thioudellet
C
C
Braun
S
S
Pavirani
A
A
D000818:Animals; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D017930:Genes, Reporter; D006801:Humans; D007536:Isomerism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D018485:Muscle Fibers, Skeletal / Q000166:cytology*; D010957:Plasmids; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives* / Q000378:metabolism; D014162:Transfection / Q000235:genetics / Q000379:methods*; D014407:Tumor Cells, Cultured
D002412:Cations; D008081:Liposomes; D013096:Spermine; D004247:DNA; D002784:Cholesterol; D008156:Luciferases; D013095:Spermidine