Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering

Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast desig...

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Autores principales: Beltran-Sastre, Violeta, Benisty, Hannah, Burnier, Julia, Berger, Imre, Serrano, Luis, Kiel, Christina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661934/
https://www.ncbi.nlm.nih.gov/pubmed/26612112
http://dx.doi.org/10.1038/srep17432
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author Beltran-Sastre, Violeta
Benisty, Hannah
Burnier, Julia
Berger, Imre
Serrano, Luis
Kiel, Christina
author_facet Beltran-Sastre, Violeta
Benisty, Hannah
Burnier, Julia
Berger, Imre
Serrano, Luis
Kiel, Christina
author_sort Beltran-Sastre, Violeta
collection PubMed
description Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce ‘TEMTAC’, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2.
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spelling pubmed-46619342015-12-01 Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering Beltran-Sastre, Violeta Benisty, Hannah Burnier, Julia Berger, Imre Serrano, Luis Kiel, Christina Sci Rep Article Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce ‘TEMTAC’, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2. Nature Publishing Group 2015-11-27 /pmc/articles/PMC4661934/ /pubmed/26612112 http://dx.doi.org/10.1038/srep17432 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Beltran-Sastre, Violeta
Benisty, Hannah
Burnier, Julia
Berger, Imre
Serrano, Luis
Kiel, Christina
Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
title Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
title_full Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
title_fullStr Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
title_full_unstemmed Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
title_short Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
title_sort tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661934/
https://www.ncbi.nlm.nih.gov/pubmed/26612112
http://dx.doi.org/10.1038/srep17432
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