CELL BIOLOGY OF THE NEURON Polarity ... - Tavernarakis Lab

Cell Biology of the Neuron: Polarity, Plasticity and Regeneration, Crete 2011
Development of a New Strategy to Control Protein
Function in the Developing Mouse CNS
Rachel Jackson 1 , Wenlin An 1 , Laura Ward 1 , Michiel Van Diepen 1 , Luke Whiley
2 2 3 4 1
, Cristina Legido-Quigley , Thomas Wandless , Karen Liu , Britta Eickholt
1
MRC Centre for Developmental Neurobiology, King's College London
2
Pharmaceutical Science Division, King's College London
3
Department of Chemical and Systems Biology, Stanford University
4
Department of Craniofacial Development, King's College London
To study a protein in vivo it is desirable to perturb its normal function in a
spatially and temporally restricted fashion. We are exploiting a recently
developed approach to achieve this, based on a strategy that allows protein
function to be regulated in a rapid, reversible and tuneable manner. In this
technique, a destabilising domain derived from E. coli dihydrofolate reductase
(DHFR*) is fused to a protein of interest, causing its efficient degradation.
Presence of a DHFR* ligand, the antimicrobial drug trimethoprim (TMP),
stabilises the protein and confers biological activity. TMP is a cost-effective drug
widely used in veterinary and medical applications, which passes the blood-brain
barrier making it an ideal tool to manipulate proteins in the CNS.
We combine in utero electroporation of DHFR* tagged constructs into the cortex
of mouse embryos with subsequent stabilisation by systemic application of TMP.
We have tested methods to deliver the synthetic ligand, analysing its ability to
cross the blood-brain barrier, and investigated if DHFR* fusion proteins can be
stabilised rapidly and reversibly. These characteristics will introduce a new layer
of control to inducible gene expression systems currently used in mice.
Using this technique we aim to study the interaction of two pathways with
multiple roles in brain development, the Semaphorins and PTEN. We have
generated DHFR*-Semaphorin ligands and DHFR*-Cre, which will be used in
combination with floxed-PTEN alleles. This system allows for combinatorial
control of two or more DHFR* fusion proteins, enabling the investigation of
possible interactions between these two pathways in mice.
Presented by: Jackson, Rachel
Poster No 052
Red Session
134