Immunological separation of rat precartilaginons stem cells and construction of pTRE-PTHrP(38-94) responsive plasmids

Objective To establish the method of separating and purifying rat precartilaginous stem cells (PCSCs) by immunomagnetic technology, clone the mid-fragment of parathroid hormonerelated peptide gene PTHrP(38-94) and construct plasmids of tetracycline(Tet) responsive element which regulates and controls the expression of PTHrP(38-94). Methods Immunornagnetic separation was used to segregate PCSCs labeled with fibroblast growth factor receptor-3(FGFR-3). The PCSCs were identified by imrnunocytochemistry with anti-FGFR-3 and anti-PCNA. The total RNA was extracted from PCSCs after identification and the mid-fragment of PTHrP(38-94) gene was obtained by RT-PCR method. With the added endonuclease Sites, PTHrP(38-94) gene was double-enzyme digested by Bam H Ⅰ and Not Ⅰ. The gene was subcloned to plasmids of Tet-responsive element with the selection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic expressive plasmid pTRE-PTHrP(38-94). Then the recombinant plasmids were transferred into E. coli-DH5α and the clones were randomly selected. Then the recombinant plasmids were purified and identified by doubleenzyme digestion. Results The results of immunocytochemistry confirmed that the rat pre-cartilaginous stem cells was obtained by the micro-segregate and purified by immunomagnetic technology. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasmids. Conclusions The purified PCSCs can be identified accurately and the responsive plasmids containing PTHrp(38-94) gene can be successfully constructed, which may give new strategy for investigation of the biological function of the PTHrp(38-94) gene in the differentiation of chondrocytes and osteocytes. Key words: Stem cells; Immunomagnetic separation;  Parathyroid hormone-related protein