[Adeno-associated virus mediated delivery and expression of GDNF cDNA in cultured bovine iris pigment epithelial cells].
OBJECTIVE
To obtain transgenic bovine iris pigment epithelial cells (IPECs) by adeno-associated virus (AAV) mediated delivery of cDNA of glial cell-line derived neurotrophic factor (GDNF).


METHODS
AAV-GDNF was titrated by slot blotting. Cultured bovine passage two IPECs were transfected using AAV-GDNF at dosage of MOI (multiplicity of infection) = 50, then were cultured in DMEM medium complemented with 3% FBS for 4 weeks with no change of medium. The expression of GDNF in culture medium was examined using ELISA test. By using the same methods, cultured passage two IPECs were transfected with AAV-GFP (green fluorescent protein) at dosage of MOI = 50, and then were cultured in DMEM medium complemented with 20% FBS. The expression of GFP in IPECs was examined using fluorescence microscope every 2 days after transfection.


RESULTS
(1) GFP expression in IPECs could not be detected until 4 days after transfection. The positive GFP expression in IPECs reached fastigium in day 8 or 10. (2) According to the results of ELISA test, concentration of GDNF in the culture medium was (17.14 +/- 1.10) micro g/L.


CONCLUSIONS
AAV-GDNF can effectively transfect cultured IPECs, and the transgenic cells show a high expression of GDNF.
Li
Yi-bin
YB
Beijing Institute of Ophthalmology, Beijing Tongren Ophthalmic Center, Capital University of Medical Science, Beijing 100005, China. yibinlee@hotmail.com
Sun
Bao-chen
BC
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D051100:Glial Cell Line-Derived Neurotrophic Factor; D049452:Green Fluorescent Proteins; D006801:Humans; D007498:Iris / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009414:Nerve Growth Factors / Q000235:genetics*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D018076:DNA, Complementary; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D049452:Green Fluorescent Proteins