Construction of an immortalized rat astrocyte strain with inducible enkephalin expression / 中华麻醉学杂志

ABSTRACT

Objective To construct an immortalized rat astrocyte strain expressing enkephalin which can be regulated by doxycycline.Methods pRevTet-On and recombinant plasmid pRevTRE/hPPE were transfected into packaging cell PT67 respectively by standard lipofectamine methods.The supernatant containing RevTet-On virus was used to infect immortalized rat astrocyte strain(IAST)and RevTRE/hPPE virus was used to infect the IAST/Tet-On cell.Immortalized rat astrocyte strain that stably expressed Tet-regulated enkephalin was established. hPPE gene expression level of IAST/Tet-On/hPPE cell strain was detected by real time-PCR,immuno- cytochemistry and radio-immunoassay.Results Real time-PCR,immuno-cytochemistry and radio-immunoassay confirmed the level of enkephalin expression was regulated by doxycycline in a dose-dependent manner.Conclusion An immortalized rat astrocyte strain which secrets enkephalin as controlled by doxycycline has been constructed successfully.