Construction of an immortalized rat astrocyte strain with inducible enkephalin expression

AIM: To construct an immortalized rat astrocyte strain (IAST) expressing enkephalin regulated by Doxycycline. METHODS: Human preproenkephalin gene (hPPE) was inserted into the plasmid pRevTRE to construct recombinant retroviral vector pRevTRE/hPPE. pRevTet-On and pRevTRE/hPPE were separately transfected into packaging cell PT67. The RevTet-On virus supernatants were used to infect IAST and positive colonies were transiently transfected with pRevTRE-Luc plasmid. One IAST/Tet-On clone with high inducibility and low background was selected by detecting luciferase activity. RevTRE/hPPE virus was used to infect the IAST/Tet-On strain. IAST that stably expressed Tet-regulated enkephalin was established. hPPE gene expression levels of IAST/Tet-On/hPPE cell strains were detected by RT-PCR, immunocytochemistry and indirect immunofluorescence staining. RESULTS: A recombinant retroviral vector pRevTRE/hPPE was constructed successfully. The inducibility multiple of the IAST/Tet-On cell strain was 20.6. RT-PCR and immunocytochemistry confirmed that enkephalin expression level was regulated by Doxycycline in a dose-dependent manner. CONCLUSION: An IAST which secretes enkephalin under the control of Doxycycline has been established successfully, which provides a good basis for further research on regulated gene therapy for chronic pain.