title: Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology.
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.
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Dale O
authors.initials: DO
authors.lastname: Cowley
authors.affiliation: TransViragen, Inc., Research Triangle Park, NC 27709, USA.
authors.forename: Debra
authors.initials: D
authors.lastname: Banner
authors.affiliation: Animal Models Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Holle
authors.affiliation: Key Lab of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Liguo
authors.initials: L
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Lishan
authors.initials: L
authors.lastname: Su
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004622:Embryo, Mammalian / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005260:Female; D005821:Genetic Techniques; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques / Q000379:methods*; D053631:Interleukin Receptor Common gamma Subunit / Q000172:deficiency / Q000235:genetics; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D008969:Molecular Sequence Data; D011247:Pregnancy; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; C507942:Il2rg protein, mouse; D053631:Interleukin Receptor Common gamma Subunit; D000094704:RNA, Guide, CRISPR-Cas Systems; D006867:Hydrolases; C020156:fumarylacetoacetase