Regulated expression of a gene of interest is crucial for transgenic research, as well as for safe and efficacious gene therapy. The most commonly used conditional expression system requires the generation of two transgenic strains, one carrying an inducible promoter and the other a transactivator. The generation of conditional transgenic models using this method is costly and time consuming. In this study, we report the design and construction of novel simplified gene delivery vectors that integrate both the regulatory and responsive elements in a single vector. The Tet-On system was used and integrated the inducible promoter and transactivator in a single plasmid, between which a copy of an insulator was inserted to minimize interference between the two units. Another copy of an insulator was inserted upstream of the transgene cassette to eliminate transgene silencing and to lower basal expression. The two insulators were in the same orientation. To further decrease basal expression, the most powerful repressor domain containing a ‘kruppel-associated box’ of the zinc finger protein NK10 was used and fused to TetR in one of the two vectors. The function of this system was confirmed after in?vitro transient transfection. The two conditional plasmids were successfully constructed, and the expression of the gene of interest was regulated tightly in vitro. In conclusion, the vectors described here may be useful for gene therapy applications, as well as for the establishment of conditional animal models.

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