It's extremely weird. In my current research, my gene X is essential for cell survive. I knockout gene X in the cells (CreERT2; X flox/flox) by treating 1uM 4-OHT for 24h. So X will depleted very well, and the cell will die at day 5 post treatment. In the following experiment, I delivery exogenous flag-tagged X into the cells using lentivirus (plasmid: plvx3-X ), and the expression of flag-X was validated by WB. Then I add 4-OHT into the medium to knockout the endogenous gene, however, the cells still die.
here is the group:
1. Ctrl (plvx3), cell survive;
2. Ctrl (plvx3) + 4-OHT, cell die;
3. plvx3-X + 4-OHT, cell die.
I cann't figure out why? plvx3-X construction was sequenced. Exogenous X protein was also expressed, the level even more higher than the endogenous one, why it didn't work?
the Flag tag affect its function? but many papers also use flag-tagged X.
Or the toxicity of activated CreERT2 ? 1uM 4-OHT was added for 24h, then the medium was replaced.
Could anyone help me figure it out ? Be greatly appreciated !
- Badges
- Science topic