[Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene].
OBJECTIVE
To clone and eukaryotic express wild type and truncated mouse ciliary neurotrophic factor (CNTF) gene, and to observe the biological effect of two types of CNTF gene expressing in ARPE-19 cells.


METHODS
RT-PCR was used to amplify the cDNA of CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected to ARPE-19 cells. Dot blotting was used to detect the expression of CNTF. MTT and flow cytometry apoptosis assay were performed to observe the biological effect of CNTF expressing in ARPE-19 cells.


RESULTS
Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasmids were successfully constructed. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF were detected in the supernatant of cells culture. MTT result shows that two types of CNTF have no proliferation promoting effect to ARPE-19 cells, and quantitive apoptosis assay implicated that CNTF could partially suppress the apoptosis that induced by the cells culturing with serum free culture.


CONCLUSIONS
Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.
Yuan
Song-tao
ST
Tianjin Medical University, Tianjin 300070, China.
Li
Ning-dong
ND
Zuo
Ai-jun
AJ
Liang
Dong-chun
DC
Zhao
Kan-xing
KX
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors; D051379:Mice; D008810:Mice, Inbred C57BL; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D020934:Ciliary Neurotrophic Factor