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Switching On and Off Transgene Expression within Lactotrophic Cells in the Anterior Pituitary Gland in Vivo¹

Molecular Medicine and Gene Therapy Unit, University of Manchester School of Medicine (J.R.S.-A., J.C.W., D.S., P.R.L., M.G.C.), Manchester, United Kingdom M13 9PT; and AstraZeneca (J.S.), Macclesfield, Cheshire, United Kingdom SK10 4TG

Address all correspondence and requests for reprints to: Prof. M. G. Castro or Prof. P. R. Lowenstein, Molecular Medicine and Gene Therapy Unit, Room 1.302, Stepford Building, University of Manchester School of Medicine, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: mcastro@fs1.scg.man.ac.uk or lowenstein{at}man.ac.uk

To further develop our understanding of anterior pituitary (AP) function and to aid the development of gene therapy strategies for the treatment of pituitary diseases, adenovirus (Ad)-mediated gene transfer to the AP gland will be a useful tool. Although successful widespread gene transfer within the AP has been achieved using first generation Ads the ability to control transgene expression would be very beneficial when studying AP regulatory functions and delivering a potentially therapeutic gene into the AP gland. A dual adenoviral vector system encoding for cell type-specific and regulatable transcription units was developed to achieve transcriptionally targeted transgenesis within specific cell populations in the adult AP gland. To achieve regulatable transgene expression within predetermined AP cells, the tetracycline-responsive transcriptional elements have been engineered to be under the control of human, lactotroph-specific PRL (hPRL) promoter elements within a dual adenoviral vector system. The inducibility, cell type specificity, and levels of transgene expression were characterized in vitro and in vivo and compared with the strong ubiquitous ß-actin/human cytomegalovirus (CAG) promoter. Inducible expression of the marker gene ß-galactosidase under the control of the hPRL promoter was restricted to lactotrophic tumor cell lines and lactotrophic cells within primary AP cultures. Lactotroph cell type specificity and inducible transgene expression were also observed within the AP gland in vivo, and this could be switched on or off. Administration of doxycycline abrogated transgene expression both in vitro and in vivo. Our results also provide evidence that an excess of trans-activator is needed to achieve maximal transgene expression. Our data indicate that combined transcriptional and inducible transgenesis can be achieved using adenoviral vectors that allow spatial and temporal restriction of transgene expression within the adult AP gland in vivo.

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