期刊
NATURE PROTOCOLS
卷 9, 期 8, 页码 1825-1847出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.103
关键词
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资金
- Jane Coffin Childs Memorial Fund for Medical Research
- University of California, San Francisco Program for Breakthrough Biomedical Research
- Leukemia and Lymphoma Society
- Howard Hughes Medical Institute, National Institutes of Health [1U01CA168370-01]
- Howard Hughes Collaborative Initiative Award
Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and for defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of 'hit' genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each round of screening can be implemented in similar to 2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and we present complete experimental procedures, as well as a full computational analysis suite for the identification of hits in pooled screens and generation of genetic interaction maps. Although the protocol outlined here was developed for our original shRNA-based approach, it can be applied more generally, including to CRISPR-based approaches.
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