Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells.
OBJECTIVE
To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro.


METHODS
A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery.


RESULTS
Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected.


CONCLUSIONS
Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.
Abul-Hassan
K
K
University Department of Ophthalmology, Manchester Royal Eye Hospital, Manchester, United Kingdom.
Walmsley
R
R
Boulton
M
M
D000368:Aged; D002130:Calcium Phosphates / Q000633:toxicity; D002411:Cation Exchange Resins / Q000008:administration & dosage; D003637:DEAE-Dextran / Q000633:toxicity; D004247:DNA / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008055:Lipids / Q000008:administration & dosage; D008081:Liposomes; D008156:Luciferases / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008875:Middle Aged; D010714:Phosphatidylethanolamines / Q000008:administration & dosage; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000008:administration & dosage; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D008164:Luminescent Proteins; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; C485838:calcium phosphate, monobasic, anhydrous; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D003637:DEAE-Dextran; C020243:calcium phosphate; D008156:Luciferases; C485829:calcium phosphate, dibasic, anhydrous