Identification of epiphyseal proliferating zone cells and construction of eukaryotic expression vector containing PTHrp gene

Objective To separates proliferating zone cells from the growth plate,then clone the parathroid hormone-related peptide gene (PTHrp) and construct its eukaryotic expression vector.Method After centrifugation through a discontinuous Percoll gradient,the third and fourth fractions cell populations of growth plate chondrocytes were identified with anti-collagen type X and electron microscope image.The total RNA was extracted from cells after identification and the full length eDNA encoding PTHrp gene was obtained by RT-PCR method and inserted into pCR2.1 TA cloning vector.After the sequencing was confirmed,the gene was subcloned to pEGFP-IRES_2 to construct recombinant eukaryotic expression vector pEGFP-IRES_2-PTHrp.Result The proliferating zone cells were separated from growth plate.Enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant vector.Conclusion The proliferating zone cells were identified accurately and the eukaryotic expression plasmid containing PTHrp gene was successfully constructed,which may be a promising for studying the biological function of the PTHrp gene and its role in chondrocyte differentiation and bone formation.