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A modified Tet-ON system minimizing leaky expression for cell-type specific gene induction in medaka fish
Osamu Hosoya1, Myung Chung2,3, Satoshi Ansai4
1Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Development, Growth & Differentiation
|August 10, 2021
Summary
Researchers developed a Tet-ON system for inducible gene expression in medaka fish rod photoreceptors. This modified system significantly reduced transgene leakiness, enabling precise temporal and spatial control.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- The Tet-ON system is crucial for inducible gene expression.
- Controlling gene expression temporally and spatially is vital in research.
- Existing Tet-ON systems may exhibit leakiness, limiting their precision.
Purpose of the Study:
- To develop a modified Tet-ON system for inducible transgene expression in medaka fish rod photoreceptors.
- To improve the spatiotemporal control of gene expression in a specific cell type.
- To reduce transgene leakiness in the Tet-ON system for medaka fish.
Main Methods:
- Generation of a transgenic medaka fish line using a modified reverse tetracycline-controlled transcriptional transactivator (rtTAm).
- Construction of a self-reporting vector with rtTAm driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) linked to GFP.
- Administration of doxycycline to larval fish to assess transgene induction and localization.
Main Results:
- The modified rtTAm significantly reduced transgene leakiness in medaka fish.
- Green fluorescent protein (GFP) expression was successfully restricted to rod photoreceptors.
- GFP fluorescence intensity showed dose-dependent and time-dependent enhancement with doxycycline treatment (up to 72 hours).
Conclusions:
- The developed Tet-ON system with modified rtTAm provides effective spatiotemporal control of transgene expression in medaka fish rod photoreceptors.
- This system offers a valuable tool for studying gene function in specific cell types within living medaka.
- The improved Tet-ON system enhances the precision of inducible gene expression studies in zebrafish models.
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