title: A dual molecular analogue tuner for dissecting protein function in mammalian cells.
abstract: Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and particularly in stem cells, are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways, and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid, reversible, titratable and independent manner. By engineering molecular tuners for NANOG, CHK1, p53 and NOTCH1 in mammalian stem cells, we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes.
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, 13-20E, New York, New York 10029, USA.
authors.forename: Ran
authors.identifier: 0000-0001-9714-8623
authors.initials: R
authors.lastname: Brosh
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, 13-20E, New York, New York 10029, USA.
authors.forename: Iryna
authors.initials: I
authors.lastname: Hrynyk
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, 13-20E, New York, New York 10029, USA.
authors.forename: Jessalyn
authors.initials: J
authors.lastname: Shen
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, 13-20E, New York, New York 10029, USA.
authors.forename: Avinash
authors.initials: A
authors.lastname: Waghray
authors.affiliation: Department of Pharmacology and Howard Hughes Medical Institute, University of Washington, Box 357280, Seattle, Washington 98195, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zheng
authors.affiliation: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.|Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, 13-20E, New York, New York 10029, USA.|The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.|Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.
authors.forename: Ihor R
authors.initials: IR
authors.lastname: Lemischka
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1 / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D010937:Plant Growth Regulators / Q000494:pharmacology; D034622:RNA Interference; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D000071317:Nanog Homeobox Protein; D010937:Plant Growth Regulators; D051881:Receptor, Notch1; D016159:Tumor Suppressor Protein p53; D000071877:Checkpoint Kinase 1