Summer Scolarship Project; Arkell Lab

 

Project: Is the post-translational addition of a SUMO moiety required for Zic5 function during embryonic development?

 

 

Project Background and Aim: The members of the Zic gene family, to which Zic5 belongs, are critical mediators of embryonic development. Mutations in the Zic genes are associated with a range of congenital birth defects in humans. Despite their importance to embryonic development, there is still little information as to how the proteins encoded by the Zic genes function and are regulated. Therefore, one of the main aims of our lab is to understand the molecular mechanism(s) underlying Zic protein function and regulation. To address our aims, we use in vivo mouse models and a range of in vitro techniques. This summer project is based on a mouse strain that carries a mutation in Zic5 (Zic5R) which stops a post-translational modification of the Zic5 protein and disrupts the function of this protein. This project aims to identify the type of post-translational modification that is disrupted by the mutation. This will be achieved by expressing the wild-type and mutant Zic5 proteins in mammalian cells and determining whether it co-localises with known modifiers such as the small polypeptide, SUMO.

 

Experimental Approach:

To examine whether SUMO and Zic5 co-localise in cells, you will:

·   Make a plasmid construct that ‘tags’ SUMO with a myc epitope that can be recognized by commercially available anti-myc antibodies.

·   Grow HEK 293T cells and co-express the myc-tagged SUMO construct with either the wild-type or mutant Zic5 proteins in these cells.

·   Use fluorescently labeled anti-Zic5 and anti-myc antibodies to detect Zic5 and SUMO, respectively, in these cells.

·   Use confocal microscopy and image analysis software to determine whether wild-type Zic5 and SUMO are co-localised and whether this co-localisation is disrupted in the mutant Zic5 protein.

 

In this project, you will learn the following molecular and cell biology techniques:

·        PCR

·        Cloning using a recombineering method

·        Tissue culture and transfection of mammalian cells

·        Preparation of cells for immunofluorescence microscopy

·        Confocal microscopy and Image Analysis.