부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.gene.2005.08.019 Byline: Iciar MuA[+ or -]oz, Manuel Carrillo, Silvia Zanuy, Ana Gomez Keywords: Inducible system; tTA transactivator; rtTA transactivator; Gene expression Abbreviations: CMV, strong immediate early promoter of cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; Dox, doxycycline; EPC, carp epithelioma papulosum cell line; FBS, foetal bovine serum; GFP, green fluorescent protein; hsp70, heat-shock protein 70; LUC, luciferase; PBS, phosphate buffer saline; RLU, relative light units; rtTA, reverse tetracycline-regulated transactivator; Tc, tetracycline; tTA, tetracycline-regulated transactivator Abstract: The exogenous control of foreign gene expression is relevant to both basic research and biotechnological applications. In fish, the number of isolated genes has become larger in the last few years; however an efficient system for controlling gene expression is not yet available. The tetracycline-regulated system has proved to be efficient and it is widely used in mammals, but it has never been tested in fish. This work includes the establishment of the tetracycline-regulated system for use in fish cells, and the determination of the optimal conditions to achieve a tight exogenous expression regulation. We have compared the tet-off and tet-on systems and the performance of the transactivators under the control of promoters with different origin and strength. The results show that the tet-off is more efficient than the tet-on system for use in fish cells. The hCMV promoter/enhancer proved to be more efficient than the carp [beta]-actin promoter to drive the expression of the transactivator, since the use of the carp [beta]-actin promoter resulted in a high intra-clonal variability when stably expressed. An auto-regulated system approach proved useful only when transiently expressed. Author Affiliation: Department of Fish Reproductive Physiology, Instituto de Acuicultura de Torrelasal, CSIC, Ribera de Cabanes, 12595, Torrelasal, Castellon, Spain Article History: Received 7 June 2005; Revised 9 August 2005; Accepted 12 August 2005 Article Note: (miscellaneous) Received by M. Schartl