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

Samenvatting

Source: Berens, C. et al. Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling. J. Vis. Exp. (2015)

This video demonstrates the use of an inducible expression system to identify where the bacterial effector protein CaeB disrupts the host apoptotic cascade. It outlines the steps for transfecting CaeB-expressing cells with a tetracycline-responsive system to induce pro-apoptotic gene expression in order to identify the point of disruption within the cascade.

Protocol

1. Transfection of the Stable Cell Line with the Inducible Expression Vector System

1. Seed human embryonic kidney (HEK)293 cells stably expressing green fluorescent protein (GFP) or GFP-CaeB in a 12-well plate at a density of 1 x 10⁵ cells/well.
2. 19 hr post-seeding, co-transfect the cells with the regulator plasmid and response plasmid, either encoding Bax or activated caspase 3.
   1. Co-transfect the stable HEK293 cells with 100 ng of pWHE125-P regulator plasmid (Figure 1) and 100 ng of the response plasmids pWHE655-hBax or pWHE655-revCasp3 (Figure 2) and 0.4 µl of polyethylenimine.
   2. Prepare the deoxyribonucleic acid (DNA) and polyethylenimine transfection reagent each in 75 µl of Opti-MEM medium and incubate for exactly 5 min at room temperature (RT). For complex formation, incubate both mixtures together for 15 min at RT.
3. Add the polyethylenimine/DNA solution dropwise to the cells and incubate at 37 °C in 5% CO₂.

2. Induction of Apoptosis

1. 5 hr post-transfection, induce expression of the pro-apoptotic proteins by the addition of 1 µg/ml doxycycline to the culture medium. Incubate cells for 18 hr at 37 °C in 5% CO₂.

Resultaten

Figure 1. Schematic overview of the regulatory plasmid pWHE125. Elements essential for propagation in ...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
DMEM	Life technologies	31966-021
FCS	Biochrom	S0115
Pen/Strep	Life technologies	15140-122
OptiMEM	Life technologies	51985
X-tremeGENE 9	Roche	6365752001
Polyethylenimine	Polyscienes	23966
Doxycycline	Sigma Aldrich	D9891