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

K. Abul-Hassan, R. Walmsley, M. Boulton

Research output: Contribution to journalArticle

39 Citations (Scopus)

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 successfully achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

Original languageEnglish (US)
Pages (from-to)361-366
Number of pages6
JournalCurrent Eye Research
Volume20
Issue number5
StatePublished - 2000
Externally publishedYes

Fingerprint

Retinal Pigments
Liposomes
Epithelial Cells
Transfection
Genes
DEAE-Dextran
Firefly Luciferases
DNA
Green Fluorescent Proteins
Luciferases
Complementary DNA

Keywords

  • Cationic liposomes
  • Gene transfer
  • GFP
  • Luciferase
  • Non-viral vectors
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Abul-Hassan, K., Walmsley, R., & Boulton, M. (2000). Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells. Current Eye Research, 20(5), 361-366.

Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells. / Abul-Hassan, K.; Walmsley, R.; Boulton, M.

In: Current Eye Research, Vol. 20, No. 5, 2000, p. 361-366.

Research output: Contribution to journalArticle

Abul-Hassan, K, Walmsley, R & Boulton, M 2000, 'Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells', Current Eye Research, vol. 20, no. 5, pp. 361-366.
Abul-Hassan, K. ; Walmsley, R. ; Boulton, M. / Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells. In: Current Eye Research. 2000 ; Vol. 20, No. 5. pp. 361-366.
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N2 - 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 successfully achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

AB - 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 successfully achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

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