Construction of Immortalized Precartilaginous Stem Cells from Neonate Rats by Transfection with Simian Virus 40 T Antigene Gene

Objective To establish immortalized precartilaginous stem cells(PSCs) from neonatal SD rats in vitro for the further related research on the differentiation mechanism and clinic application of precartilaginous stem cells. Methods By using LipofectamineTM2000,a gene transfection reagent,plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigene gene(SV40Tag) was transfected into the primary cultured PSCs isolated by immuniomagnetic beads coasted with the second antibody.Colonies were isolated by puromycin selection and expanded by many passages.Investigate the capability of differentiation of the transfected cells.The expression of SV40Tag in expanded cell lines was identified by immunocytochemistry method and RT-PCR. Results A particular anti-puromycin cell clone was acquired,which was confirmed as fibroblast growth factor receptor-3(FGFR-3) positive PSCs.The total RNA were isolated from the positive cell clones,and a 588 bp fragment,which was specific for the SV40T antigene gene,was amplified.The transfected cells were expanded to immortalized cell strain,named as immortalized precartilaginous stem cells(IPSCs).The population doubling time of IPSCs was (22.98±2.77) h,no significant effect of subculture,freezing and recovering had been found. Conclusion Precartilaginous stem cells could be isolated from neonatal SD rats,cultured in vitro,and immortalized through the transfection of pCMVSV40T/PUR.