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Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation
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Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector.

Aisha Yesbolatova1,2, Yuichiro Saito1, Masato T Kanemaki1,2

  • 1Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka 411-8540, Japan.

Pharmaceuticals (Basel, Switzerland)
|May 28, 2020
PubMed
Summary

We simplified the auxin-inducible degron (AID) system for mammalian cells using bicistronic plasmids. This method enables efficient depletion of target proteins like MCM10, aiding the study of protein function and conditional mutant generation.

Keywords:
auxin-inducible degronconditional protein depletionexpression vectorgene knockout

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Conditional degron systems allow rapid protein depletion upon ligand addition, aiding protein function studies.
  • The auxin-inducible degron (AID) system, while popular, is complex due to its two-component nature (OsTIR1 and mAID).
  • Existing methods like dTAG and HaloPROTAC offer alternatives, highlighting the need for simplified AID applications.

Purpose of the Study:

  • To simplify the auxin-inducible degron (AID) system for use in mammalian cells.
  • To develop a streamlined method for conditional protein depletion using bicistronic plasmids.
  • To create a tool for generating conditional mutants by combining simplified AID with CRISPR technology.

Main Methods:

  • Construction of bicistronic all-in-one plasmids expressing OsTIR1 and mAID-fused proteins via a P2A self-cleavage sequence.
  • Generation of a HeLa mutant line for the replication factor MCM10 using CRISPR-knockout and the bicistronic plasmid.
  • Depletion of mAID-MCM10 protein upon addition of indole-3-acetic acid (auxin).

Main Results:

  • Successfully developed simplified, bicistronic all-in-one plasmids for AID system components in mammalian cells.
  • Established HeLa mutant lines exhibiting auxin-inducible depletion of mAID-fused MCM10.
  • Demonstrated the efficacy of the simplified AID system for controlling transgene-derived protein levels.

Conclusions:

  • The developed bicistronic plasmids offer a simplified and efficient approach to the AID system in mammalian cells.
  • This technology facilitates the construction of conditional mutants by integrating with CRISPR-based gene knockout strategies.
  • The simplified AID system provides a valuable tool for studying protein function and genetic regulation.