pRetroX-PTuner载体描述 pRetroX-PTuner is a bicistronic, retroviral expression vector that allows you to precisely regulate the amount of your protein of interest in stably transduced mammalian cells. The vector encodes a 12 kDa, FKBP (L106P) destabilization domain (DD; 1) that is expressed as an N-terminal tag on your protein of interest; this domain causes the rapid degradation of any protein to which it is fused. Once expressed, the amount of DD-tagged protein present in the cell can be rapidly increased by the addition of Shield1 stabilizing ligand to the medium. Shield1 is a membrane permeable molecule that binds to the DD tag, 'shielding' the fusion protein from proteasomal degradation. pRetroX-PTuner allows the simultaneous expression of your DD-tagged protein of interest and puromycin acetyltransferase (PAC; for puromycin resistance (Puror)) from the same bicistronic mRNA transcript. Because PAC (i.e., Puror) is unaffected by the DD tag, it can be used as a selection marker.
Bicistronic expression of PAC and the DD-tagged protein of interest is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). This IRES allows cap-independent translation of PAC (Puror) from an internal start site at the IRES/Purorjunction (2). A gene cloned into the multiple cloning site (MCS), located between the DD and the IRES sequences, is expressed as a bicistronic message transcribed from the 5’ LTR.
pRetroX-PTuner is derived from the pMIN series of retroviral vectors (3, 4). These optimized vectors have the ability to produce high viral titers, and high levels of recombinant protein. In addition, because they lack retroviral structural genes (gag, pol, and env) necessary for retroviral particle formation and replication, these vectors exhibit improved safety profiles.
pRetroX-PTuner contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, the packaging signal (Ψ), and the tRNA primer-binding site. pRetroX-PTuner also contains a ColE1 origin of replication, and an E. coli Ampr gene for propagation and selection in bacteria
pRetroX-PTuner is available in the Retro-X ProteoTuner Shield System N . It is designed to efficiently deliver and co-express your DD-tagged protein of interest and puromycin acetyltransferase (Puror) in any mitotically active mammalian cell. In order to create your DD-tagged protein of interest, your gene of interest must be cloned into the MCS in the same reading frame as the DD tag sequence, and it must contain a stop codon at the end of its coding sequence.
In order to infect mammalian cells with pRetroX-PTuner, the vector must be transfected into a packaging cell line, such as the RetroPack PT67 Cell line, AmphoPack-293, EcoPack2-293 , Pantropic Retroviral Expression System , or Retro-X Universal Packaging System . These cell lines package RNA transcribed from the vector into infectious, replication-incompetent, retroviral particles. Such retroviral particles can transduce target cells and transmit the gene of interest, but cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chance of producing replication-competent virus due to recombination events during cell proliferation.
When cells expressing a DD-tagged protein of interest are grown in medium containing Shield1, the ligand binds to the DD tag and protects the fusion protein from degradation. As a result, the protein quickly accumulates inside the cells in amounts that are directly proportional to the concentration of Shield1 in the medium. If the cells are subsequently grown in medium lacking Shield1, the DD tag is no longer stabilized, and the fusion protein is rapidly degraded. Because the effects of Shield1 are concentration-dependent
and reversible, it is possible to fine-tune the amount of fusion protein present in the cells simply by adjusting the concentration of Shield1 in the medium (1). Propagation in E. coli Suitable host strains: Stellar Competent Cells.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: ColE1
Copy number: high
