Induction of shRNAmir (tracked by TurboRFP) with doxycycline is tightly regulated

HEK293T cells were transduced at an MOI of 0.3, puromycin selected (2ug/ml) for 96 hours after which 500ng/ml of doxycycline (Dox) was added to the cells and TurboRFP expression was assessed at 48-72hrs (B,C). Post-Dox samples (D,E) were split 1:8, washed with PBS and photographed at times indicated indicated. At 72 hrs after Dox removal TurboRFP expression is reduced to background levels.

shRNAmir expression is not leaky in the absence of doxycycline

Residual tetracycline in media does not result in leaky expression. The pTRIPZ vector does not express shRNAmir to GAPDH at levels capable of producing significant knockdown, in the absence of doxycyline (whether in media with residual tetracycline or in special tetracycline free media). Each bar represents four tissue culture replicates all triplicated in QPCR for a total of 12 data points each. Statistical analysis was performed and no statistically significant differences were found between nonsilencing and GAPDH shRNAmir samples. Transduction at an MOI =5 was performed in HEK293T cells after which cells were selected with 2ug/ml puromycin for 96hrs.

Tet-On or Tet-Off configurations

The pTRIPZ vector has been engineered to provide for induced expression of an shRNAmir in the presence of doxycycline (Limited Use License - Tet Technology). There are two main components on the pTRIPZ vector enabling induction:

(1) The tetracycline response element (TRE) and
(2) The transactivator.

The TRE, modified from its natural state to consist of a string of operators fused to the CMV minimal promoter, exhibits reduced basal expression and tighter binding to the second component, the transactivator. The pTRIPZ transactivator, known as the reverse tetracycline transactivator 3 (rtTA3) binds to and activates expression from TRE promoters in the presence of doxycycline. The rtTA3 transactivator is a modified version of the wildtype in two ways. First, unlike the original tetracycline transactivator the rtTA3 is modified to bind to the TRE in the presence of doxycycline rather than in its absence. Secondly, there are three mutations within the transactivator that increase its sensitivity to doxycycline by 25 fold over the initial rtTA without increasing background activity (Das, et al. 2004).

The pTRIPZ vector is versatile in that it can be easily converted to a Tet-Off® capable vector using Cre/loxP technology or classical restriction digest. The rtTA3 is flanked by loxP sites allowing in vitro or in vivo excision of the rtTA3 by exposure to Cre recombinase. The rtTA3 is also flanked by a pair or BamHI restriction sites allowing for straightforward cleavage and ligation of the vector to remove the rtTA3. Without the rtTA3 present on the vector a tetracycline transactivator (tTA) can be added extraneously to the system allowing it to function as Tet-Off; where expression of shRNAmir and TurboRFP are alternatively induced in the absence of doxycycline.

See product insert for additional details