University of Cambridge > Talks.cam > BSS Formal Seminars > Proteomic and cell biological approaches to the study of clathrin in a vertebrate conditional knockout cell-line

Proteomic and cell biological approaches to the study of clathrin in a vertebrate conditional knockout cell-line

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In eukaryotic cells, clathrin is required for the selective transport of proteins between intracellular membrane compartments. To understand the role of clathrin in vivo, we exploited the unique properties of the chicken B cell-line DT40 . This cell-line has become established as an important tool in cell biology because it exhibits a remarkably high rate of homologous recombination, thus allowing targeted gene knockouts to be easily generated. We replaced endogenous clathrin genes in DT40 with a clathrin gene under the control of a regulatable promoter. Hence cells can be grown either with clathrin or without and their properties compared. Using a whole cell proteomic methodology called LOPIT (Localisation of Organelle Proteins by Isotope Tagging) we have completed the first proteomic analysis of the major organelles in DT40 and compared the changes that occur when clathrin expression is repressed. In this talk I will explain how the LOPIT methodology works and will show how the use of DT40 has produced new insights into clathrin function.

This talk is part of the BSS Formal Seminars series.

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