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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.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.issn 2045-2322
dc.identifier.uri http://hdl.handle.net/10230/57357
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.language.iso eng
dc.publisher Nature Research
dc.relation.ispartof Scientific Reports. 2015 Nov 27;5(1):17432
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.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.identifier.doi http://dx.doi.org/10.1038/srep17432
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.accessRights info:eu-repo/semantics/openAccess
dc.type.version info:eu-repo/semantics/publishedVersion


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