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.
Highlights
Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research
Transgenic manipulation of NRG and NSG mice through embryo microinjection is difficult due to the poor embryo yields produced through natural mating[14,32]
Most current NOD transgenic and NOD knockout mice were derived by backcrossing the mutant genes or transgenes from other mouse strains
Summary
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. The ability to introduce additional mutations directly in embryos from NRG or NSG mice would greatly facilitate further improvements in humanized mouse models Novel approaches, such as zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas[9] nucleases, have greatly accelerated the production of genetically modified mice using direct injection of DNA or mRNA into one-cell stage embryos[21,22,23,24,25], allowing construction of knockout mice by directly manipulating a small number of embryos
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