Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering

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• dc.contributor.author Beltran-Sastre, Violeta
• dc.contributor.author Benisty, Hannah, 1986-
• dc.contributor.author Burnier, Julia
• dc.contributor.author Berger, Imre
• dc.contributor.author Serrano Pubull, Luis, 1982-
• dc.contributor.author Kiel, Christina
• dc.date.accessioned 2023-06-27T06:18:59Z
• dc.date.available 2023-06-27T06:18:59Z
• dc.date.issued 2015
• dc.description Includes supplementary materials for the online appendix.
• dc.description.abstract 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.
• dc.description.sponsorship We thank all members of our laboratories for their contributions and helpful discussions. We thank the CRG Genomics Unit and the Biomolecular Screening & Protein Technologies Unit. We acknowledge help in the quantifications of the Western blots by Dina Cramer and assistance in RNA sequencing analysis by Javier Delgado. Armelle Yart provided the HEK293 cells stably expressing GH receptor. This work was funded by the European Commission (EC) Framework programme (FP) 7 projects PRIMES (contract nr. 278568), ComplexINC (contract nr. 279039) and SynSignal (contract nr. 613879). LS is supported by the Spanish Ministerio de Economía y Competitividad, Plan Nacional BIO2012-39754 and the European Fund for Economic and Regional Development. We are particularly grateful for the support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017′ (SEV-2012-0208).
• dc.format.mimetype application/pdf
• dc.identifier.citation Beltran-Sastre V, Benisty H, Burnier J, Berger I, Serrano L, Kiel C. Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering. Sci Rep. 2015 Nov 27;5(1):17432. DOI: 10.1038/srep17432
• dc.identifier.doi http://dx.doi.org/10.1038/srep17432
• dc.identifier.issn 2045-2322
• dc.identifier.uri http://hdl.handle.net/10230/57357
• dc.language.iso eng
• dc.publisher Nature Research
• dc.relation.ispartof Scientific Reports. 2015 Nov 27;5(1):17432
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/278568
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/279039
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/613879
• dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/BIO2012-39754
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/SEV-2012-0208
• dc.rights 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/
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.other Malalties
• dc.subject.other Proteïnes
• dc.subject.other Anomalies cromosòmiques
• dc.subject.other Mutació (Biologia)
• dc.title Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion