Question

Outline an in vitro experiment you could carry out to show that Arnt (Aryl hydrocarbon Receptor Nuclear Translocator) is essential for the dioxin receptor to bind to the response element of the Cytochrome P450 1A1 gene.

Answer 1

The experiment can be processed in following manner:

1. Generate a cell-line by genetic engineering and knock-out technology which lacks the Arnt domain.
2. Generate two separate cell-culture systems, each having equal number of cells in similar growth conditions.
3. Induce the culture systems with stimulators which activate cytochrome P4501A1 gene.
4. Since one of the cell culture system lacks Arnt now, only one of the cell system will be able to internalize the stimulant now.
5. After stimulation, scrape the cells and prepare nuclear lysates.
6. Analyse both the cell lysates for expressin of cytochrome P4501A1 by western blotting.
7. The cell type which expresses the gene cytochrome P4501A1 will show that the Arnt is necessarily required for its activation while the stimulant binds to the nuclear membrane-associated dioxin receptors.