1. Proteome-scale tagging and functional screening in mammalian cells by ORFtag
- Author
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Nemčko, Filip, Himmelsbach, Moritz, Loubiere, Vincent, Yelagandula, Ramesh, Pagani, Michaela, Fasching, Nina, Brennecke, Julius, Elling, Ulrich, Stark, Alexander, and Ameres, Stefan L.
- Abstract
The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.
- Published
- 2024
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