Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells.

Abstract:

PURPOSE: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.

journal_name

Curr Eye Res

journal_title

Current eye research

authors

Abul-Hassan K,Walmsley R,Boulton M

subject

Has Abstract

pub_date

2000-05-01 00:00:00

pages

361-6

issue

5

eissn

0271-3683

issn

1460-2202

journal_volume

20

pub_type

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