Standardization of procedure for efficient ex vivo gene transfer into porcine pancreatic islets with cationic liposomes.
BACKGROUND
New strategies to improve the outcome of encapsulated porcine islet transplantation may involve the transfer of gene sequences affecting islet viability. While adenoviral vectors appear as the most efficient gene transfer system so far established for islets, non-viral-based vectors are most likely to fulfill microbiological safety criteria and be retained in the clinical setting. Our aim was to standardize the procedures of gene transfer into adult porcine islets using cationic liposome DOTAP.


METHODS
Porcine islets obtained by collagenase digestion and density gradient purification were lipofected with plasmids coding for luciferase or beta-galactosidase under the control of simian virus 40 or cytomegalovirus promoter. The following parameters were explored: exposure time to vector (1-48 hr), DNA amount (1-15 microg/500 islets), and DOTAP to DNA ratio (2-16). Reporter gene expression was determined 48-72 hr after lipofection.


RESULTS
Efficiency and reproducibility of transfection were maximal with the following procedure: 3-hr exposure time followed by islet washing, 12 microg of DNA per 500 islets (150 microm equivalent), and DOTAP to DNA ratio of 12 microl/microg. Freshly isolated islets in large aliquots (n=4000 in 50-ml tubes) were efficiently transduced with this procedure, and distribution of gene expression was homogenous when islets were subsequently plated in 500-islet aliquots. Luciferase gene expression was detected for a minimum of 7 days after lipofection. Gene expression was also evident up to 4 weeks after islet transplantation beneath the kidney capsule of athymic mice. Transfection of islets using the beta-galactosidase vector yielded 25% positive islets. Islet viability was not adversely affected.


CONCLUSIONS
This islet lipofection procedure may help achieve the local release of a bioactive peptide in the graft environment and have therapeutic applications in islet transplantation.
Benhamou
P Y
PY
Department of Endocrinology, University Hospital, Grenoble, France. benhamou@ujf-grenoble.fr
Moriscot
C
C
Prevost
P
P
Rolland
E
E
Halimi
S
S
Chroboczek
J
J
D000818:Animals; D002412:Cations; D002470:Cell Survival; D004247:DNA / Q000032:analysis; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D007515:Islets of Langerhans / Q000166:cytology; D008081:Liposomes; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives; D013552:Swine; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C098223:2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate; D002412:Cations; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D013096:Spermine; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane