Using DT40 to study clathrin function.

Abstract

Clathrin-coated pits and vesicles play a major role in eukaryotic membrane trafficking pathways. We have used the DT40 system to delete endogenous clathrin genes selectively from DT40 and replace them with clathrin under the control of a tetracycline-regulatable promoter. This enabled clathrin expression to be manipulated, and the functional consequences of clathrin depletion to be studied in a stable vertebrate context. Here we describe the background to the work on clathrin, our practical experience with using the tetracycline-regulatable expression system in DT40 and some novel insights into membrane trafficking pathways obtained using the cell-lines generated during the course of this work.