title: Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering.
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.
authors.affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Violeta
authors.initials: V
authors.lastname: Beltran-Sastre
authors.affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Hannah
authors.initials: H
authors.lastname: Benisty
authors.affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Julia
authors.initials: J
authors.lastname: Burnier
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, B.P. 181, Grenoble, France.|The School of Biochemistry, University of Bristol, Clifton BS8 1TD, United Kingdom.
authors.forename: Imre
authors.initials: I
authors.lastname: Berger
authors.affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluís Companys 23, 08010 Barcelona, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
authors.affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Christina
authors.initials: C
authors.lastname: Kiel
mesh_terms: D000818:Animals; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D008156:Luciferases; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)