Related Experiment Video
Updated: Jul 16, 2026
Functional Cloning Using a Xenopus Oocyte Expression System
Published on: January 30, 2016
[Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene]
Song-tao Yuan1, Ning-dong Li, Ai-jun Zuo
1Tianjin Medical University, Tianjin 300070, China.
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.
Conclusion:
Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.
