The beneficial optical properties of zebrafish allow not only passive observation, but also active manipulation of proteins and cells through light using optogenetic tools. Initially, photosensitive ion channels have been applied to the neurobiology of zebrafish to analyze complex behavior at the cellular level. The strict regulation of protein localization in cell compartments is very important for many biological processes, and its imbalance can lead to diseases. Studying these processes by manipulating protein localization requires tools that can be fast and have high spatial resolution. Several photogenetic zebrafish strain frameworks have been constructed to shuttle proteins between specific subcellular compartments or form clusters between each other.
Figure: Gene expression and protein visualization (Kardash, 2012)Creative BioMart has been able to provide our customers with zebrafish models adapted to optogenetic tools to study protein localization. At present, there are three kinds of optical switches that can be used to regulate protein localization, Phytochrome B, Cryptochrome 2 and Improved Light-Inducible Dimer (iLID). Based on the above switching system, we can provide the following zebrafish strains.
In many biological processes, the strict regulation of intracellular protein localization is very important, and its imbalance can lead to diseases. To study these processes by manipulating protein localization requires fast and high spatial resolution tools.
Mechanism of action: Light-induced heterodimer, stable in dark-state, disruption upon>740nm light illumination.
Application in zebrafish: Gene expression in a luciferase assay, sub-cellular protein re-localization
The photoreactive Arabidopsis proteins phytochrome B (PHYB) and bHLH transcription factor phytochrome interaction partner (PIF) heterodimerized under red light irradiation in the presence of chromophore phycocyanin (PCB). After activation, the interaction remained stable in the dark for several hours. In addition, far red illumination dissociates the dimer. PIF3 has nuclear localization signal, which is used to transfer this chimeric transcription factor to the nucleus of zebrafish under red light irradiation, so as to control the expression of luciferase. Activation and binding occurred within a few seconds, and the dissociation kinetics was less than a minute after far red light irradiation.
Mechanism of action: Light-induced homooligomerization.
Cry2olig is a protein aggregation system. When stimulated by blue light, CRY2 will self aggregate, which helps to induce protein relocation. Cry2olig is expected to become a general tool to study protein aggregation related diseases or control protein activity by delocalization.
Mechanism of action: Light-induced heterodimer, disruption in dark-state.
Application in zebrafish: Induction of mitophagy by protein re-localization.
The system is composed of E. coli peptides SspB and SRA fused to Avena sativa LOV2 domain. Under blue light stimulation, the affinity of their binding partners changes greatly. The iLID system provides rapid activation and deactivation mechanics and lacks homomonomer. iLID was applied to zebrafish to regulate mitotic phagocytosis.
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