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Including the p53 ELAV-like protein-binding site in vector cassettes enhances transgene expression in rat
1Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-1190, USA. czheng@dir.nidcr.nih.gov
Objective:
ELAV-like proteins regulate mRNA stability and/or translation. We evaluated whether inclusion of binding sites for ELAV-like HuR proteins in vector cassettes could improve transgene expression in the salivary gland.
Methods:
Western blots and immunofluorescence staining were used to determine whether HuR protein was expressed in salivary cells and tissue. HuR binding sites were inserted into the pACEF1α-luc-BGH expression plasmid. Cell lines were transfected with plasmids in vitro and luciferase expression measured. Rat submandibular glands were transfected in vivo with plasmids containing ELAV-like HuR protein-binding sites. An adenoviral vector with p53 ELAV-like HuR protein-binding site was generated and also tested in vivo. Four unique 29mer HuR shRNA constructs were used in A5 cells to evaluate whether there was a specific interaction between HuR protein and the p53 HuR protein-binding site.
Results:
Salivary cells express HuR protein. Inclusion of the p53 ELAV-like HuR protein-binding site resulted in high luciferase activity in salivary cells in vitro, with similar results in vivo. In vitro shRNA data demonstrated that the high luciferase activity was mediated by the interaction between HuR protein and the p53 HuR protein-binding site. The AdEF1α-luc-p53BGH, including this binding site, mediated very high luciferase activity, ~4-fold that seen with the CMV promoter, in rat submandibular glands.
Conclusions:
Including the p53 ELAV-like protein-binding site in transgene cassettes may enhance therapeutic vectors intended for use with salivary glands.
