Wisconsin/Project/BCL2
From 2007.igem.org
Contents |
BCL-2 and Cell Fate
Background
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate bcl-2 (a pro-survival protein) and prevent cells from dying under mild stress. We have designed an inducible artificial transcription factor (ATF) that binds to bcl-2 promoter. Currently we are studying cell fate regulation using ATF in mouse fibroblast cells.
Design
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself.
Experiment
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.
