Cloning PTHrP sub-genes and construction of eukaryotic responsive plasmids in rats

Objective: To clone rat parathyroid hormone-related peptide(PTHrP) sub-genes and construct the plasmids of the tetracycline(Tet) responsive element,which may regulate and control the expressions of the PTHrP sub-genes.Methods: Total RNA was extracted from precartilaginous stem cells and the PTHrP(1-36),PTHrP(38-94) and PTHrP(107-139) sub-genes were obtained by the RT-PCR method.Then the sub-genes were subcloned into the plasmids of the Tet-responsive element with the selection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic responsive plasmids.After transferred into E.coli-DH5α,the clones were amplified and the recombinant plasmids purified and identified by double-enzyme digestion.Results: Double enzyme digestion analysis and sequencing showed that the target sub-genes were cloned into the recombinant plasmids.Conclusion: The eukaryotic responsive plasmids containing the PTHrP(1-36),PTHrP(38-94) and PTHrP(107-139) sub-genes were successfully constructed,which might contribute a lot to the rigorous regulation of the expressions of PTHrP sub-genes.