期刊
PLOS ONE
卷 13, 期 6, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0198790
关键词
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资金
- Presidential Early Career Award for Scientists and Engineers
- Department of Defense BCMRP Era of Hope Scholar Award [W81XWH-14-1-0191]
- [NIH-NCI-R01CA166284]
Gene editing protocols often require the use of a subcloning step to isolate successfully edited cells, the behavior of which is then compared to the aggregate parental population and/or other non-edited subclones. Here we demonstrate that the inherent functional heterogeneity present in many cell lines can render these populations inappropriate controls, resulting in erroneous interpretations of experimental findings. We describe a novel CRISPR/Cas9 protocol that incorporates a single-cell cloning step prior to gene editing, allowing for the generation of appropriately matched, functionally equivalent control and edited cell lines. As a proof of concept, we generated matched control and osteopontin-knockout Her2(+) and Estrogen receptor-negative murine mammary carcinoma cell lines and demonstrated that the osteopontin-knockout cell lines exhibit the expected biological phenotypes, including unaffected primary tumor growth kinetics and reduced metastatic outgrowth in female FVB mice. Using these matched cell lines, we discovered that osteopontin-knockout mammary tumors were more sensitive than control tumors to chemotherapy in vivo. Our results demonstrate that heterogeneity must be considered during experimental design when utilizing gene editing protocols and provide a solution to account for it.
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