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Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector

Conditional degron-based methods are powerful for studying protein function because a degron-fused protein can be rapidly and efficiently depleted by adding a defined ligand. Auxin-inducible degron (AID) is a popular technology by which a degron-fused protein can be degraded by adding an auxin. Howe...

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Autores principales: Yesbolatova, Aisha, Saito, Yuichiro, Kanemaki, Masato T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281097/
https://www.ncbi.nlm.nih.gov/pubmed/32456235
http://dx.doi.org/10.3390/ph13050103
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author Yesbolatova, Aisha
Saito, Yuichiro
Kanemaki, Masato T.
author_facet Yesbolatova, Aisha
Saito, Yuichiro
Kanemaki, Masato T.
author_sort Yesbolatova, Aisha
collection PubMed
description Conditional degron-based methods are powerful for studying protein function because a degron-fused protein can be rapidly and efficiently depleted by adding a defined ligand. Auxin-inducible degron (AID) is a popular technology by which a degron-fused protein can be degraded by adding an auxin. However, compared with other technologies such as dTAG and HaloPROTAC, AID is complicated because of its two protein components: OsTIR1 and mAID (degron). To simplify the use of AID in mammalian cells, we constructed bicistronic all-in-one plasmids that express OsTIR1 and a mAID-fused protein using a P2A self-cleavage sequence. To generate a HeLa mutant line for the essential replication factor MCM10, we transfected a CRISPR-knockout plasmid together with a bicistronic plasmid containing mAID-fused MCM10 cDNA. After drug selection and colony isolation, we successfully isolated HeLa mutant lines, in which mAID–MCM10 was depleted by the addition of indole-3-acetic acid, a natural auxin. The bicistronic all-in-one plasmids described in this report are useful for controlling degradation of a transgene-derived protein fused with mAID. These plasmids can be used for the construction of conditional mutants by combining them with a CRISPR-based gene knockout.
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spelling pubmed-72810972020-06-15 Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector Yesbolatova, Aisha Saito, Yuichiro Kanemaki, Masato T. Pharmaceuticals (Basel) Article Conditional degron-based methods are powerful for studying protein function because a degron-fused protein can be rapidly and efficiently depleted by adding a defined ligand. Auxin-inducible degron (AID) is a popular technology by which a degron-fused protein can be degraded by adding an auxin. However, compared with other technologies such as dTAG and HaloPROTAC, AID is complicated because of its two protein components: OsTIR1 and mAID (degron). To simplify the use of AID in mammalian cells, we constructed bicistronic all-in-one plasmids that express OsTIR1 and a mAID-fused protein using a P2A self-cleavage sequence. To generate a HeLa mutant line for the essential replication factor MCM10, we transfected a CRISPR-knockout plasmid together with a bicistronic plasmid containing mAID-fused MCM10 cDNA. After drug selection and colony isolation, we successfully isolated HeLa mutant lines, in which mAID–MCM10 was depleted by the addition of indole-3-acetic acid, a natural auxin. The bicistronic all-in-one plasmids described in this report are useful for controlling degradation of a transgene-derived protein fused with mAID. These plasmids can be used for the construction of conditional mutants by combining them with a CRISPR-based gene knockout. MDPI 2020-05-23 /pmc/articles/PMC7281097/ /pubmed/32456235 http://dx.doi.org/10.3390/ph13050103 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yesbolatova, Aisha
Saito, Yuichiro
Kanemaki, Masato T.
Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
title Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
title_full Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
title_fullStr Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
title_full_unstemmed Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
title_short Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector
title_sort constructing auxin-inducible degron mutants using an all-in-one vector
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281097/
https://www.ncbi.nlm.nih.gov/pubmed/32456235
http://dx.doi.org/10.3390/ph13050103
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