Development of new T-vectors containing the luciferase gene. Easy application for direct cloning of a promoter DNA.

Abstract

For promoter analyses of genes, it is usually necessary to amplify promoter DNA fragments by polymerase chain reaction (PCR) and clone them into a plasmid containing a reporter gene. In the present study we developed a novel plasmid, pGL2-X, which was constructed through a simple procedure of cloning an XcmI cassette from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene into the multicloning site of pGL2-Basic (Promega) pGL2-X was then converted by XcmI digestion into a T-vector which was named pGL2-T. Unfortunately, however, the firefly luciferase gene in pGL2-Basic contains one XcmI restriction site and therefore one base within the recognition site was silent-mutated. The cloning efficiency of the pGL2-T vector was approximately 63% when tested with a PCR product amplified from a promoter region (-501(-)+24) of the murine acetylcholine receptor delta subunit (AchR delta) gene. In C2C12 muscle cells transiently transfected with pGL2-T containing the AchR delta promoter, transcription of the silent-mutated luciferase gene increased 2.2-fold by neuregulin (EGF domain of heregulin beta 1; 100 ng/mL), a known stimulator of AchR delta expression. This result suggested that the pGL2-T vector was biologically functional. Thus, the present study provides an easy method to construct a variety of T-vectors containing different reporter genes.