Objective: To construct the pEGFP-C1-Sam68 eukaryotic expression vector and establish a cell line stably expressing pEGFP-C 1-Sam68.Methods: Sam68 amplified by PCR,and identified by electrophoresis analysis.The PCR products and the eukaryotic Expression vector pEGFP-C1 were digested by restriction endonuclesaes,then the digested Sam68 was inserted into the digested eukaryotic Expression Vector.After screening,the positive recombinants were picked out.PCR analysis,Restriction endonuclesaes digest analysis and sequence analysis were used to identify the recombinants.pEGFP-C 1-Sam68 was transfected into PC12 cells with lipofectamine to obtain a cellline stably expressing pEGFP-C 1-Sam68 Results: The 1320 bp DNA fragment was amplified by PCR,the pEGFP-C 1-Sam68 eukaryotic expression vector was successfully constructed and the cell line stably expressing pEGFP-C1-Sam68 was established.Real-time PCR and Western blotting revealed a 394-fold pEGFP-C 1-Sam68 overexpression in the transfected PC 12 cells as compared with that in control PC 12 cells transfected with pEGFP-C 1 (P<0.01).Conclusion: The protocol can be used to construct the cell line stably expressing pEGFP-C 1-Sam68 to provide a cell model for studying alternative splicing about Sam68.