pRetroX-Tet-Off Advanced载体描述 pRetroX-Tet-Off Advanced is a Retro-X Q retroviral vector that expresses tTA-Advanced, an improved version of the tetracycline(Tet)-controlled transactivator protein tTA (1-4). The tTA-Advanced protein is a fusion of the Tet repressor (TetR) DNA-binding domain, and three minimal “F”-type transcriptional activation domains from the herpes simplex virus VP16 protein. The gene encoding tTA-Advanced is completely synthetic, lacks cryptic splice sites, and utilizes human codon preferences for stable expression in mammalian cells. Expression of tTA-Advanced is driven by the powerful, constitutively active CMV promoter.
As with all of our Retro-X Q vectors, pRetroX-Tet-Off Advanced uses LTR self-inactivation to eliminate promoter interference. In LTR self-inactivation, the mechanism of viral integration is used to introduce a deletion into the 5’ LTR. As a result of this mutation, the 5’ LTR CMV/ MSV promoter, which drives high expression of the complete viral genome in packaging cells, is inactive in stably transduced target cells. This allows the expression of tTA-Advanced (from PCMV) to proceed unimpeded (5, 6). The vector also contains a neomycin resistance gene (Neor) that allows G418 selection of stably transduced cells. tTA-Advanced and the Neor marker are coexpressed from a bicistronic transcript containing an internal ribosome entry site (IRES). This ensures that a high frequency of G418 resistant clones express the Tet-Off Advanced transactivator.
pRetroX-Tet-Off Advanced contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, a packaging signal (Ψ+), and a tRNA primer binding site. The vector also contains an SV40 origin of replication for plasmid propagation in mammalian cells that express SV40 T antigen, as well as a ColE1 origin of replication and an E. coli Ampr gene for propagation and selection in bacteria.
The pRetroX-Tet-Off Advanced vector is used in conjunction with Tet response vectors to create double stable, Retro-X Tet-Off Advanced cell lines. Such cell lines are essentially Doxycycline (Dox)-controlled gene expression systems in which the Tet response vector expresses a gene of interest under the control of a Tet-responsive element (TRE; e.g. PTight or PTRE2; 7). In the absence of Dox, tTA-Advanced binds to the TRE and activates expression of the gene of interest. In the presence of Dox, tTA-Advanced is unable to bind to the TRE, and the system is inactive. Additional information on TRE-containing vectors, and protocols describing how to construct a Retro-X Tet-Off Advanced cell line can be found in the Retro-X Tet-Off Advanced Inducible Gene Expression System User manual (PT3959-1). Selection of Stable Transfectants Selectable marker: plasmid confers resistance to G418 (neomycin). Propagation in E. coli Suitable host strains: DH5α, DH10B and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: ColE1
Copy number: high
