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  • Title: Pooled Generation of Lentiviral Tetracycline-Regulated microRNA Embedded Short Hairpin RNA Libraries.
    Author: Adams FF, Hoffmann T, Zuber J, Heckl D, Schambach A, Schwarzer A.
    Journal: Hum Gene Ther Methods; 2018 Feb; 29(1):16-29. PubMed ID: 29325442.
    Abstract:
    Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.
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