Transfection of basic fibroblast growth factor (bFGF) gene or bFGF antisense fene into human retinal pigment epithelial cells.
BACKGROUND
Transplantation of RPE cells offers a potential of restoring retinal pigment epithelium (RPE) function and has been shown to be effective in the dystrophic RCS rat model. Recently, RPE transplantation was attempted in patients with age-related macular degeneration. Basic fibroblast growth factor (bFGF) plays important roles in maintaining normal retinal function. The purpose of this study was to introduce bFGF sense or antisense cDNA into human RPE cells to alter the expression of bFGF.


METHODS
Human bFGF sense cDNA or antisense cDNA was inserted into the pBK-CMV vector. For stable gene expression, we introduced the plasmids into RPE cells using the electroporation method. Following electroporation, transfected RPE cells were cultured and resistant cells were selected in the presence of antibiotic G418. We analyzed the expression of the transfected genes in the cloned RPE cells by polymerase chain reaction (PCR) and by reverse transcription (RT)-PCR.


RESULTS
Cloned RPE cells in which the bFGF sense or antisense cDNA had been efficiently transfected were established. PCR and RT-PCR analysis demonstrated not only the presence but also the expression of bFGF sense or antisense cDNA in the transfected RPE cells.


CONCLUSIONS
Human bFGF sense cDNA or antisense cDNA can be efficiently introduced into cultured RPE cells by the electroporation method. The successful expression of the genes into RPE cells demonstrated that this technique can be used to regulate bFGF expression and thus increase the scope of RPE transplantation for the treatment of retinal diseases.
Ogata
N
N
Department of Ophthalmology, Kansai Medical University, Moriguchi, Osaka, Japan. ogata@takii.kmu.ac.jp
Nishizawa
M
M
Ando
A
A
Uyama
M
M
D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D016373:DNA, Antisense / Q000235:genetics* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D016373:DNA, Antisense; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2