Control of red fluorescent protein expression by tetracycline in an adenoviral bivectorial system

RODRIGUEZ SS; REGGIANI PC; SCHWERDT JI; RIMOLDI OJ ; GOYA RG.

Puerto Madryn

Congreso; XLVI Reuni¨®n Anual de la SAIB; 2010

Sociedad Argentina de Investigaci¨®n Bioqu¨ªmica

We built a tetracycline-responsive system (Tet-Off) consisting of  two recombinant adenoviral vectors (RAds), one harbornig a  sequence coding for a tetracycline-responsive transactivator  protein, and another coding for the transgene for the red fluorescent protein DsRed2 under control of a minimal promoter containing the tetracycline-response element. We characterized the functionality of the system in cell cultures which were co-transduced with both RAds. In order to assess vector s regulatability, cells were cultured with doxycycline (DOX) 1¦Ìg/ml. High expression of DsRed2 was observed (red fluorescence) in cells grown without DOX, but only minimal fluorescence levels were detected in cells treated with DOX. In addition, to study the system performance in vivo, rats were allotted to 2 groups which received stereotaxic injections of both RAds in the brain. One group received regular tap water (Control group) whereas the other one received water containing 1mg/ml DOX (DOX group). On day 2 post injection, brains were removed and coronal sections prepared. Transgene expression was assessed by fluorescence microscopy. In the control but not in the DOX group, numerous DsRed2 fluorescent cells were observed. We conclude that our Tet-Off system is suitable for implementing gene delivery to the brain and subsequently regulating transgene expression by administration or removal of DOX.