Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System
Transcripción
Begin by taking a suspension of cultured recipient cells in a tube. Supplement the tube with inducible plasmids containing the gene of interest. The expression of this gene is under the control of an upstream promoter sequence fused to a Tetracycline operator or TetO, forming a tetracycline-responsive element or TRE.
Additionally, add regulatory tetracycline-off vectors, each containing a gene encoding the recombinant tetracycline transactivator protein or tTA. Electroporate the cell-DNA mix to use electrical pulses to facilitate the uptake of plasmids by the cell.
Post-electroporation, plate the cells and incubate for the desired time duration. Upon entering the nucleus, the tetracycline-off vector expresses tTA protein. The tTA is a hybrid protein containing a Tet repressor or the TetR moiety and a viral VP16 transcription transactivator domain.
TetR moiety binds the TetO sequence of the TRE element, while the VP16 domain allows the RNA polymerase to synthesize the mRNA from the downstream DNA, eventually leading to gene expression. Replace the growth media in the culture plate with doxycycline-containing media and incubate.
Doxycycline – a synthetic tetracycline analog – binds to the TetR protein and changes its conformation. This change disrupts the interaction of TetR protein with the TetO sequence, inhibiting the target gene expression.
