High efficiency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipid-DNA complex.
Efficient transfection conditions for a number of human, rat and rabbit primary cells and established lines of vascular origin have been determined using a complex of a commercially available cationic lipid transfection agent (Tfx-50) and luciferase reporter plasmid constructs. The optimised conditions have also been successfully applied to rabbit carotid arteries in vivo and a series of human arteries in vitro. The most critical factors influencing the efficiency of gene transfection with this protocol are: DNA concentration; ratio of lipid reagent to DNA; transfection time and the presence or absence of serum. Immunohistochemical analysis shows that a high percentage of cells (approximately 30-80% dependent on lineage) were transfected under optimal conditions with minimal toxicity effects. Similar analyses performed on undamaged rabbit carotid vessels transfected in vivo and human arteries transfected in vitro show high-efficiency transfer and strong expression of the luciferase vector as demonstrated by reporter gene expression. The optimisation of gene transfer into vascular cells with this cationic lipid complex will be valuable for molecular studies of genes implicated in cardiovascular diseases and as a possible method of gene delivery with therapeutic intent.
Keogh
M C
MC
Thrombosis Research Institute, London, UK.
Chen
D
D
Lupu
F
F
Shaper
N
N
Schmitt
J F
JF
Kakkar
V V
VV
Lemoine
N R
NR
D000818:Animals; D001808:Blood Vessels / Q000166:cytology* / Q000201:enzymology; D017536:Carotid Artery, Common / Q000201:enzymology; D018929:Cell Culture Techniques; D004247:DNA / Q000008:administration & dosage*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D015316:Genetic Therapy; D006801:Humans; D008055:Lipids; D008081:Liposomes; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D014162:Transfection
D008055:Lipids; D008081:Liposomes; D004247:DNA; D008156:Luciferases