In vitro and in vivo optimization of Destabilizing Domain in neuron for modeling brain disease
- Title
- In vitro and in vivo optimization of Destabilizing Domain in neuron for modeling brain disease
- Authors
- BAEK, SEUNG TAE; 이희은; 곽민준; 김예은
- Date Issued
- 2021-05-20
- Publisher
- 한국뇌신경화학회
- 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.
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/109777
- Article Type
- Conference
- Citation
- KSBNS 2021, 2021-05-20
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