The pSingle-tTS-shRNA vector expresses the tetracyline-controlled transcriptional suppressor (tTS), which controls expression of an shRNA sequence inserted into the shRNA cloning site. The tTS protein is a fusion of the Tet repressor protein (TetR) and the KRAB-AB silencing domain of the Kid-1 protein (SDKid-1 ), a powerful transcriptional suppressor (1, 2). In the absence of the inducer doxycycline (Dox, a tetracycline derivative), tTS binds to the tetO sequences in the modified Tet-responsive Pol III hybrid promoter (P Tight/U6 ) and blocks expression of the shRNA. As Dox is added to the culture medium, tTS dissociates from P Tight/U6 to allow Pol IIImediated transcription of the shRNA, resulting in suppression of the target gene in a highly dose-dependent manner. pSingle-tTS-shRNA also contains a ColE1 origin of replication and an ampicillin resistance gene (Amp r ) for propagation and selection in bacteria, as well as a neomycin resistance gene (Neo r ) for selection of stable transformants in mammalian cells.

载体应用

After digesting pSingle-tTS-shRNA with HindIIIand XhoI, clone an annealed ds oligonucleotide encoding your desired shRNA sequence into the vector. Transfect the recombinant vector into cells and select stable, clonal tranformants with G418 to obtain a cell line capable of induced suppression of the shRNA's target gene.