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Sci Rep. 2014 Jun 17;4:5290. doi: 10.1038/srep05290.

Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology.

Author information

1
Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
2
1] Animal Models Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA [2] TransViragen, Inc., Research Triangle Park, NC 27709, USA.
3
TransViragen, Inc., Research Triangle Park, NC 27709, USA.
4
Animal Models Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
5
Key Lab of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
6
1] Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA [2] Key Lab of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [3] Department of translational medicine, Department of surgery, Department of medicine, the first hospital, Jilin University, Changchun 130061, China.

Abstract

Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research. Immunodeficient mice such as NOD-Rag1-/-IL2RgammaC-null (NRG) or NOD-SCID-IL2RgammaC-null (NSG) mice are critical for efficient engraftment of human cells or tissues. However, their genetic modification remains challenging due to a lack of embryonic stem cells and difficulty in the collection of timed embryos after superovulation. Here, we report the generation of gene knockout NRG mice by combining in vitro fertilization (IVF) and CRISPR/Cas9 technology. Sufficient numbers of fertilized embryos were produced through IVF, and a high rate of Fah gene targeting was achieved with microinjection of Cas9 mRNA, gRNA and single strand oligonucleotide DNA (ssDNA) into the embryos. The technology paves the way to construct NRG or NSG mutant mice to facilitate new humanized mouse models. The technology can also be readily adapted to introduce mutations in other species such as swine and non-human primates.

PMID:
24936832
PMCID:
PMC4894429
DOI:
10.1038/srep05290
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Yes, there is potential Competing Interest. D. Cowley is employed by, has equity ownership in and serves on the board of directors of TransViragen, the company which has been contracted by UNC-Chapel Hill to manage its Animal Models Core Facility. No other authors have any competing financial interests.

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