Project description:Determining protein function in a systematic and comprehensive manner is a key goal in modern molecular biology research but is challenging with current approaches. Here, we introduce ORFtag, a versatile method for the massively parallel protein tagging and interrogation of protein function at the proteome scale with minimal effort and expense. We demonstrate the versatility of ORFtag in mouse and human cells with a series of functional screens for transcriptional activators, repressors and post-transcriptional gene regulatory proteins.
Project description:The comprehensive assessment of protein function is a key aim in modern biology yet has proven difficult with existing approaches. Here, we introduce ORFtag, a versatile tool for the massively parallel protein tagging and functional screening at proteome scale. ORFtag integrates a cassette bearing a promoter, the coding sequence of a protein tag, and a splice-donor site, which allows the tag to be expressed and spliced to genomic protein-coding exons and thus enables endogenous protein tagging at minimal expenditure and effort. We demonstrate the versatility of ORFtag by three functional screens for transcriptional activators and repressors and mediators of mRNA translation and stability.
Project description: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 proteome scale. ORFtag utilizes retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous ORFs. We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and post-transcriptional regulators in mouse embryonic stem cells. Each screen finds known and novel regulators, including long ORFs inaccessible by other methods, revealing that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.
Project description:Determining protein function in a systematic and comprehensive manner is a key goal in modern molecular biology research but is challenging with current approaches. Here, we introduce ORFtag, a versatile method for the massively parallel protein tagging and interrogation of protein function at the proteome scale with minimal effort and expense. We demonstrate the versatility of ORFtag in mouse and human cells with a series of functional screens for transcriptional activators, repressors and post-transcriptional gene regulatory proteins.
Project description: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 proteome scale. ORFtag utilizes retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous ORFs. We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and post-transcriptional regulators in mouse embryonic stem cells. Each screen finds known and novel regulators, including long ORFs inaccessible by other methods, revealing that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.