Transfection of an Inducible Gene Expression System into Cells Expressing a Bacterial Effector Protein

Take test human cells expressing CaeB, an anti-apoptotic bacterial protein.

Use cells lacking CaeB as the control.

Introduce a transfection reagent complexed with a tetracycline-inducible gene expression system containing both regulator and response plasmids.

The regulator plasmid encodes a transcriptional activator and silencer, while the response plasmid encodes a pro-apoptotic protein controlled by TRE.

Incubate the cells to facilitate plasmid uptake and regulatory protein expression.

Without tetracycline, the activator remains inactive, and the silencer represses TRE-driven transcription.

Add a tetracycline analog and incubate.

The analog enables activator–TRE binding, initiating pro-apoptotic protein expression.

In control cells, this triggers apoptotic signaling, activating an apoptotic marker.

In CaeB-expressing cells, CaeB inhibits apoptotic signaling, reducing the marker activation.

A reduced marker activation in CaeB-expressing cells suggests that CaeB inhibits apoptosis.

Begin this procedure by seeding HEK293 cells stably expressing GFP or GFP-CaeB in a 12 well plate at a density of 100,000 cells per well.

Next, prepare DNA and polyethylenimine transfection reagent separately in 75