Plasmid map of constructed pCMV-HSVtk-P2A-EGFP and pGFAP- 3 HSVtk-P2A-EGFP 4.

(A) HSVtk (1131 bps) gene was subcloned into pEGFP-N3 at XhoI and EcoRI cutting sites. A 66 bps self-cleavage peptide P2A was also subcloned into pEGFP-N3 at EcoRI and SalI restriction enzyme cutting sites to obtain a construct named pCMV-HSVtk-P2A-EGFP. This construct could express HSVtk and EGFP as a reporter gene separately. Kanamycin/neomycin resistance gene was used for both kanamycin selection of E.coli colony and G418 sulfate selection of cell stable clone. (B) CMV promoter of pCMV-HSVtk-P2A-EGFP was substituted by 2207 bps GFAP promoter from another plasmid, pAAV-GFAP-hChR2(H134R)-mCherry, at restriction enzyme cutting sites of AseI and XhoI. HSVtk and EGFP in this clone were controlled by GFAP promoter, which is a specific promoter of glia cell. Kanamycin/neomycin resistance gene was used for both kanamycin selection of E.coli colony and G418 sulfate selection of stable clone in the further experiments. (TIF)