title: [Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene].
abstract: 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.
authors: Tianjin Medical University, Tianjin 300070, China.
authors: Song-tao
authors: ST
authors: Yuan
authors: Ning-dong
authors: ND
authors: Li
authors: Ai-jun
authors: AJ
authors: Zuo
authors: Dong-chun
authors: DC
authors: Liang
authors: Kan-xing
authors: KX
authors: Zhao
mesh_terms: 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
chemical_list: D020934:Ciliary Neurotrophic Factor