In vitro and in vivo optimization of Destabilizing Domain in neuron for modeling brain disease

Abstract

The destabilizing domain (DD) from Escherichia coli dihydrofolate reductase (ecDHFR) is a relatively small protein (158 a.a), which can conditionally control the abundance of a protein of interests (POIs) by a molecular chaperone, trimethoprim (TMP). The DD, allowed an ideal approach for reversibly and repeatedly regulating protein stability, has been used successfully to control the protein level and potentially activity. Therefore, we aim to test the usability of the DD for selectively regulating protein stability in neurons in vivo and in vitro. First, we utilized the PiggyBac (PB) transposon system for moderate and permanent expressions of candidate genes by genome integration. Using in utero electroporation (IUE), we introduced DD-YFP to developing ventricle at embryonic day 15.5 followed by TMP administration at postnatal day 14. Strong YFP fluorescence was detected 6hr after the administration of TMP whereas minimal or no YFP fluorescence was detected in untreated group suggesting effective stabilization of DD-YFP by TMP and minimal leakage in the absence of TMP. Currently, we are generating stable human embryonic stem cell (hESC) lines expressing DD-tagged GFP followed by neuronal differentiation to validate the TMP-induced stabilization of GFP in vitro. Altogether, our study will reveal the usability of the DD for selectively regulating protein stability in neurons in vivo and in vitro.