[Construction of CNTF-GFP plasmid and transfection of Schwann cells by electroporation].
OBJECTIVE
To enhance the neurotrophic effect of Schwann cells, promote optic nerve regeneration, and simplify the means of observation, the CNTF-GFP fusion plasmid was constructed and transferred into Schwann cells by electroporation.


METHODS
It was an experimental study. Plasmid pcDNA3. 1/CNTF-GFP was constructed and verified by auto-sequence. Cultured Schwann cells were transfected by electroporation. The transfection efficiency was counted by flow cytometry, the transcription of the gene was evaluated by RT-PCR, and the expression of protein was observed by fluorescence microsphere and cell immunofluorescence.


RESULTS
CNTF-GFP plasmid was verified correctly. After electroporation, the green fluorescence of gene-transfected Schwann cells can be seen at 3 hours later, increased at 12 hours later, reached the peak during 24 h to 72 h later, and still could be seen at 7 days later. The transfection efficiency was evaluated at 44.93% +/- 12.92% by flow cytometry. The transcription of CNTF gene and the expression of CNTF protein have been proven by RT-PCR and cell immunofluorescence.


CONCLUSIONS
CNTF-GFP plasmid was constructed correctly and Schwann cells were transfected by electroporation successfully and CNTF-GFP fusion protein can be expressed well, which became a good foundation for our future's study on the transplantation of gene-modified Schwann cells to promote optic nerve regeneration.
Fang
Yuan
Y
Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.
Mo
Xiao-Fen
XF
Sun
Xing-Huai
XH
Guo
Wen-Yi
WY
Tian
Jie
J
Kong
De-Sheng
DS
Ma
Duan
D
Zhang
Meng
M
D000818:Animals; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D009416:Nerve Regeneration / Q000235:genetics / Q000502:physiology; D010957:Plasmids; D012583:Schwann Cells / Q000378:metabolism; D014162:Transfection
D020934:Ciliary Neurotrophic Factor; D049452:Green Fluorescent Proteins