The Establishment of the Reversed Immortalized Rat Dermatic Fibroblast Cell Line Induced by Cre/LoxP and SV40 LTag

Objective To explore the establishment of the reversed immortalized rat dermatic fibroblast cells.Methods The primary fibroblast cells,isolated from the newly born rat skin by collagenase digestion,were transfected with the retroviral vector SSR69 and the immortalized cells stably expressing SV40 LTag were screened.Then the immortalized cells were infected by adenovirus expressing the Cre-recombinase,and the reversed cells not expressing SV40 LTag were screened.Compared morphology,proliferation in vitro and expression characteristics between primary and immortalized fibroblast by means of inverted microscope,making growth curve and cell immunofluorescence.Tumorigenicity of reversed cells were examined by plate cloning experiment.Results The primary fibroblast cells were successfully isolated from the rat skin,and the immortalized cells were obtained by transfecting the primary fibroblast cells with the retroviral vector SSR69.Morphology of the two cells were similar,showing elongated spindle or polygonal,and grew with adherent characteristic.However,the proliferation of immortalized fibroblast was significantly superior than that of primary fibroblast(P 0.05),and the immortalized cells were cultured more than passage 25.Cell Immunofluorescence confirmed that vimentin were expressed in the cytoplasm of two cells.The reversed cells were successfully obtained after the excision of SV40 LTag gene.The proliferation of reversed cells was similar to that of primary fibroblast cells(P 0.05),and were not tumorigenic.Conclusion The reversed immortalized rat dermatic fibroblast cell line could be established by means of Cre/LoxP and SV40 LTag.