[Preparation of genetically engineered retinal pigment epithelial cells capable of secreting nerve growth factor].
OBJECTIVE
To develop genetically engineered retinal pigment epithelial (RPE) cells which are able to express bioactive nerve growth factor (NGF) steadily and efficiently and to combine gene therapy with retinal transplantation techniques to treat retinal degeneration.


METHODS
Replication-deficient retrovirus-plasmid recombinant pLXSN-NGF was constructed via subclone and clone, and then packaged into pT67 cells to obtain virion containing a secretable form of NGF gene. We evaluated titer of the retroviral recombinant with NIH 3T3 cells and then transduced NGF gene into cultured RPE cells by the retroviral recombinant. We also analyzed the expression of NGF gene in transduced and untransduced RPE cells via reverse transcription (RT)-PCR and Western blot. The bioactivity of expressed NGF were evaluated by stimulating PC 12 cells with conditioned medium from transduced and untransduced RPE cells.


RESULTS
The titer of replication-deficient retroviral recombinant reached 1.2 x 10(7) cfu/ml. NGF protein and mRNA were detected in the conditioned medium from transduced RPE cells, but not from the normal RPE cells. Marked neurite process outgrowth from PC 12 cells was observed after being stimulated with the conditioned medium from transduced RPE cells.


CONCLUSIONS
RPE cells are able to be transduced by replication-deficient retrovirus with high efficiency, and secret bioactive NGF at a high level, which might be an ideal candidate for engineered cells.
Liu
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Department of Ophthalmology, The Third Hospital, Peking University, Beijing 100083, China. liushiquan@hotmail.com
Wang
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Zhang
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H
Zhang
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Wang
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D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression; D051379:Mice; D020932:Nerve Growth Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D020932:Nerve Growth Factor