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.
Highlights
Loss-of-function studies are fundamental for dissecting gene function
We engineered pRAIDRS (RNAi and auxininduced degradation rescue system), a lentiviral vector containing all elements for construction of an auxin-regulated rescue system
A second promoter, either phosphoglycerate kinase-1 or the stronger elongation factor 1a (Supplementary Fig. 1a), followed by a Kozak sequence, drives the expression of an mRNA encoding three in-frame proteins separated by two porcine teschovirus-1 2A (P2A) peptides
Summary
Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and in stem cells, are scarce. 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 hormoneinduced degradation pathways, and is deliverable with only two lentiviral vectors. Manipulation of protein levels represents a relatively new loss-of-function approach To this end, harnessing the plant hormone-induced degradation pathways is attractive due to their efficiency and specificity. PAID has major limitations in terms of applicability to mammalian cells These include a viral promoter prone to silencing in embryonic stem cells (ESCs)[11,12], a lack of a designated selectable marker, the inability to suppress endogenous genes and a large degron (228 AAs) liable to interfere with the POI’s function. We sought to develop an improved experimental system that upgrades the stem cell biologist’s toolbox and facilitates faster, tighter and combinatorial dissection of gene and protein function
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