期刊
NATURE PROTOCOLS
卷 4, 期 12, 页码 1855-1868出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.209
关键词
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资金
- NIH [R01GM069906, R21RR024189, R21HL091808, CA118498, GM88040, AG031300]
- MGH Pathology Service
- Ned Sahin Fund
- Claflin Distinguished Scholar Award
- NATIONAL CANCER INSTITUTE [R01CA140188, R01CA118498] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR RESEARCH RESOURCES [R21RR024189] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R21HL091808] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM088040, R01GM069906] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [K01AG031300] Funding Source: NIH RePORTER
- OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH [DP1OD006862] Funding Source: NIH RePORTER
Zebrafish mutants have traditionally been obtained by using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc-finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc-Finger Consortium reagents for constructing engineered zinc-finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within 4 months.
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