A dual molecular analogue tuner for dissecting protein function in mammalian cells

Ran Brosh, Iryna Hrynyk, Jessalyn Shen, Avinash Waghray, Ning Zheng and Ihor R. Lemischka ()
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Ran Brosh: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai
Iryna Hrynyk: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai
Jessalyn Shen: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai
Avinash Waghray: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai
Ning Zheng: University of Washington
Ihor R. Lemischka: The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai

Nature Communications, 2016, vol. 7, issue 1, 1-13

Abstract: 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.

Date: 2016
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DOI: 10.1038/ncomms11742

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