Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch

The objective of this work was to further develop a tetracycline repressor (TetR) protein system that allows control of transgene expression. First, t o circumvent the need for a binary approach, a single plasmid design was co nstructed and tested in tissue culture. To indirectly assay integrations th at express the synthetic transcription factor (rtTA), a bicistronic gene wa s built which included an internal ribosome entry site (IRES) and a green f luorescent protein coding region (GFP) on the same expression cassette as t he coding region of rtTA (pTetGREEN). This construct did not produce fluore scent colonies when stably integrated and provided minimal expression of GF P in the face of adequate expression of rtTA. The coding region for TetR wa s then altered by introducing 156 silent point mutations to simulate mammal ian genes. Replacement of wild-type TetR gene (tetR) in pTetGREEN with 'mam malianized' provided GFP expression. Adjustment of codon usage in the tetR region of rtTA nearly doubled the expression level of functional rtTA. To i ncrease the number of rtTA expressing lines, the chicken egg-white lysozyme matrix attachment region (MAR) was introduced into the single plasmid desi gn just upstream of the tetracycline operators (tetO). Inclusion of the MAR doubled the number of colonies that expressed rtTA (44 vs 88). With the mo difications described here, the number of lines that express rtTA and provi de induction from a single plasmid design can be increased by the inclusion of a MAR and the level of rtTA expression can be further increased by adju sting the base composition of the TetR coding region. The MAR also insulate s the inducible gene from the promoter driving rtTA.