Construction and identification of dual expression vector pIRES2-EGFP/Cbfa1

Objective To construct the dual expression vector pIRES2-EGFP/Cbfa1 of osteoblast specific transcription factor for in vivo or in vitro research on bone.Methods Cbfa1 was amplified using PCR with pCMV/Cbfa1 containing a full length of cDNA and acting as a template,subsequently cloned with T-A,and then subcloned to the vector pIRES2-EGFP.After digestion by restriction endonucleases,PCR,and sequencing to confirm its exact sequences,pIRES2-EGFP then transfected cell NIH3T3 by liposome.After screening with G418,the total proteins were isolated.Western blot was used to detect the expression of Cbfa1.Results The exact sequences of pIRES2-EGFP/Cbfa1 vector were confirmed by digestion of restriction endonucleases,PCR,and sequencing.After transfection,the expressions of green fluorescent protein were present.The protein expression of Cbfa1 was detectable by Western blot.Conclusion The vector pIRES2-EGFP/Cbfa1 has been constructed successfully,which lays the foundation for further research.