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Sci Rep. 2015 Nov 27;5:17432. doi: 10.1038/srep17432.

Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering.

Author information

1
EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.
2
Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
3
European Molecular Biology Laboratory, Grenoble Outstation, B.P. 181, Grenoble, France.
4
The School of Biochemistry, University of Bristol, Clifton BS8 1TD, United Kingdom.
5
Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluís Companys 23, 08010 Barcelona, Spain.

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.

PMID:
26612112
PMCID:
PMC4661934
DOI:
10.1038/srep17432
[Indexed for MEDLINE]
Free PMC Article

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