Liposome-mediated transfer of genes containing HLA-class II alpha chain into cultured embryonic chick cardiac myocytes and COS7 cells.
Rejection remains a major problem after cardiac transplantation. One hypothesis is that transfer of recipient HLA genes could lead to expression of the antigens on the surface of donor cells and so facilitate graft acceptance. This paper describes a pilot study for relevant gene-transfer (transfection) experiments on adult cardiac myocytes, investigating the feasibility of transfection using cationic liposomes. The plasmid pcDV1-pL2 vector containing HLA-DR-alpha chain gene was incubated with Lipofectin, a DOTMA and DOPE lipid mixture, for 10 minutes. Embryonic chick cardiac myocytes (ECCM) and COS7 monkey cells were incubated with DNA: Lipofectin ratios of 1:1, 1:2, 1:4, and 1:10 for 16 hours (hrs). Using a fixed ratio of 1:4, incubation periods of 4, 8, and 16 hrs were compared. Finally, cells were incubated for 16 hrs and consecutively cultured for 6 days. Expression of the HLA-DR-alpha chain antigen was detected by indirect immunohistochemical staining. Highest transfection rates were achieved with a DNA: Lipofectin ratio of 1:4 for ECCM and COS7 cells (2.7% +/- 0.6% and 2.4% +/- 0.3% after 16 hrs incubation) and a transfection time of 4 hrs (5.8% +/- 0.6% and 5.3% +/- 1.7%). Immunohistochemically positive cells were present after 6 days (2.0% +/- 1.2% and 2.1% +/- 0.3%). We found a low level of expression of HLA-DR-alpha chain gene, influenced by transfection time and DNA: Lipofectin ratio. To increase the efficiency of liposome-mediated gene transfer and assess potential applications in cardiac transplantation, further investigation of cell properties promoting transfection is necessary.
Aleksic
I
I
Division of Cardiothoracic Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Ren
M
M
Czer
L S
LS
Freimark
D
D
Dalichau
H
H
Blanche
C
C
Trento
A
A
Barath
P
P
D000328:Adult; D000818:Animals; D002461:Cell Line, Transformed / Q000166:cytology; D002478:Cells, Cultured; D002642:Chick Embryo / Q000166:cytology*; D002522:Chlorocebus aethiops; D005240:Feasibility Studies; D006684:HLA-DR Antigens / Q000235:genetics*; D059805:HLA-DR alpha-Chains; D006321:Heart / Q000196:embryology*; D016027:Heart Transplantation; D006801:Humans; D007150:Immunohistochemistry; D008081:Liposomes; D009206:Myocardium / Q000166:cytology*; D010714:Phosphatidylethanolamines; D010865:Pilot Projects; D013539:Simian virus 40; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D008081:Liposomes; D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine