簡介 pcDNA5/TO is a 5.7 kb expression vector designed for use with the T-REx System (Catalog Nos. K1020-01 and K1020-02) available from Life Technologies. The vector allows tetracycline-regulated expression of the gene of interest in mammalian host cells expressing the Tet repressor (TetR) from the pcDNA6/TR vector (Catalog No. V1025-20). The T-REx System yields higher levels of induced expression than any other regulated mammalian expression system. It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known. pcDNA5/TO 載體含有以下元件: Hybrid promoter consisting of the human cytomegalovirus immediate-early (CMV) promoter and tetracycline operator 2 (TetO2) sites for high-level tetracycline-regulated expression in a wide range of mammalian cells 
 Hygromycin resistance gene for selection of stable cell lines The control plasmid, pcDNA5/TO/lacZ, is included for use as a positive control for transfection and tetracycline-regulated expression in the cell line of choice. 關于pcDNA5/TO 載體的注意事項 The pcDNA5/TO vector contains two tetracycline operator 2 (TetO2) sites within the human cytomegalovirus immediate-early (CMV) promoter for tetracyclineregulated expression of your gene of interest (Yao et al., 1998). The TetO2 sequences serve as binding sites for 4 Tet repressor molecules (comprising two Tet repressor homodimers) and confer tetracycline-responsiveness to your gene of interest. The Tet repressor is expressed from the pcDNA6/TR plasmid. For more information about the TetO2 sequences, see the next page. For more information about the pcDNA6/TR plasmid and the Tet repressor, refer to the T-REx System manual.

In the absence of tetracycline, expression of your gene of interest is repressed by the binding of Tet repressor homodimers to the TetO2 sequences. Addition of tetracycline to the cells derepresses the hybrid CMV/TetO2 promoter in pcDNA5/TO and allows expression of your gene of interest Tet 操縱子序列 The promoters of bacterial tet genes contain two types of operator sequences, O1 and O2, that serve as high affinity binding sites for the Tet repressor (Hillen and Berens, 1994; Hillen et al., 1983). Each O1 and O2 site binds to one Tet repressor homodimer. While Tet repressor homodimers bind to both tet operators with high affinity, studies have shown that the affinity of the Tet repressor homodimer for O2 is three- to five-fold higher than it is for O1 (Hillen and Berens, 1994).

Tet operators have been incorporated into heterologous eukaryotic promoters to allow tetracycline-regulated gene expression in mammalian cells (Gossen and Bujard, 1992; Yao et al., 1998). In the T-REx System, two copies of the O2 operator sequence (TetO2) were inserted into the strong CMV promoter of pcDNA5/TO to allow regulated expression of your gene of interest by tetracycline. We use the TetO2 operator sequence in pcDNA5/TO to maximize repression of basal gene expression. For more detailed information about tet operators, refer to Hillen and Berens (1994).Yao et al. (1998) have recently demonstrated that the location of tet operator sequences in relation to the TATA box of a heterologous promoter is critical to the function of the tet operator. Regulation by tetracycline is only conferred upon a heterologous promoter by proper spacing of the TetO2 sequences from the TATA box (Yao et al., 1998). For this reason, the first nucleotide of the TetO2 operator sequence has been placed 10 nucleotides after the last nucleotide of the TATA element in the CMV promoter in pcDNA5/TO. 

In other tetracycline-regulated systems, the TetO2 sequences are located upstream of the TATA element in the promoter of the inducible expression vector (Gossen and Bujard, 1992). These systems differ substantially from the T-REx System in that they use regulatory molecules composed of the Tet repressor fused to a viral transactivation domain. The presence of viral transactivation domains appears to overcome the requirement for specific positioning of the TetO2 sequences in relation to the TATA box of the heterologous promoter. However, the presence of viral transactivation domains has been found to have deleterious effects in some mammalian cell lines. T-REx 細胞系 For your convenience, Life Technologies has available several mammalian cell lines that stably express the Tet repressor. T-REx-293 cells, T-REx-HeLa cells, T-REx CHO cells, and T-REx-Jurkat cells express the Tet repressor from pcDNA6/TR and should be maintained in medium containing blasticidin. Expression of your gene of interest from pcDNA5/TO may be assayed by transfection of your pcDNA5/TO construct into any of the T-REx cell lines and induction with tetracycline.