A system for inducible protein degradation in mammalian cells.

Dr Vicki Athanasopoulos
Dr Ruth Arkell
Prof Robert Saint

Background: We learn a great deal of information about gene and protein function in cells by the selective elimination of single genes or their products. This is most commonly achieved by examining cells derived from an animal which is mutant for a gene, or by the knockdown of gene function by small double stranded RNA molecules (siRNAs). In both cases, the usual method of analysis is to wait a long enough period for the gene product to have been dramatically decreased and then study the properties of the cell. For many dynamic processes, inhibition of particular genes over a considerable length of time is likely to lead to secondary effects on cell function which may mask the primary effects that are of most interest.

The project: We recently developed a method for the induction of instability in a fusion protein in mammalian cells. The fusion protein consisted of a fluorescent protein tag linked with a target of inducible degradation. In this project, you will extend this method to induce the loss of function of a protein with a known cellular role by creating a fusion between the protein of interest and the target of the inducible degradation. You will achieve this by creating a fusion gene construct, introducing it into mammalian cells by transfection, inhibiting the normal gene function by siRNA so that the gene is dependent on the function of the fusion protein and then induce the degradation of the fusion protein. Western blot analysis will be used to examine the rate of degradation of the target while analysis of the cells will be carried out to determine the effect of depletion of the protein.

Experience gained: This project will give you experience in molecular techniques such as PCR amplification, DNA sequence analysis, construct generation, bacterial transformation, plasmid purification, mammalian cell culture, transfection of mammalian cells, microscopy, western blotting and immunohistochemical staining of cells.

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