Generation Of NOD/SCID/IL2R?^-/-/Fah^-/-?FNSG?Mouse Model

ObjectiveMouse is an organism that is widely used as a model for studying human physiology or disease progress and the development of imnunodeficient mouse with liver injury that can support either normal or cancerous human cells engraftment.Methods1.Generation of Prkdc and IL2r? gene knockout mouse with CRISPR/Cas9 system(1)Sufficient zygotes were obtained by superovulating for subsequent microinjection process;(2)The gRNA were designed for target gene knockout and obtained the mRNA by in vitro transcription;(3)Zygotes edited by microinjection and then transferred into the pseudopregnant female mouse.Waiting for neonatal mouse with target gene knockout;(4)Testing the accuracy of the knockout mouse by genotyping and sequencing;2.Generation of the NSG and FNSG strain(1)NSG strain was obtained by hybridizing NOD/Scid background mouse with homozygous of both Prkdc and IL2r? gene knockout mouse(SG)mouse.And testing the accuracy of the NSG strain mouse by genotyping and sequencing;(2)FNSG strain was obtained by hybridizing Fah background mouse with NSG strain mouse.And testing the accuracy of the NSG strain mouse by genotyping and sequencing;3.The transgenic mouse phenotype test(1)FACS analysis of single cell from the transgenic mouse of peripheral blood,bone marrow,spleen and thymus.(2)The phenotype of Fah background mouse and the FNSG strain mouse were tested by stop giving NTBC.Observing the weight variation of the mice after stop giving NTBC and obtained the slides of liver after while.Results1.The CRISPR/Cas9 system has been shown to be suitable for multiplexed genome editing.2.The Sirpa?Fah gene stable inheritance.3.Homozygous of Prkdc gene are deficient in T cells and B cells compared with heterozygous and control.Homozygous of IL2r? gene are deficient NK cells compared with heterozygous and control.The phenotype of SG mice and NSG mice.There are almost not any T cells,B cells and NK cells compared with control mouse.4.The Fah background and FNSG strain mouse lost weight after stop giving NTBC compared to wild-type mouse.The pathological sections of Fah background insight that there are severe liver injuries include the deformation of hepatic lobules,disorder of liver cell cords and massive infiltration of inflammatory cells;The pathological sections of Fah with one of imnunodeficient gene knock out mouse inform that the hepatic lobule disappears,liver cell cord scrambling severely;As for FNSG mouse,slices are eompletely loss of normal structure,sever damage and necrosis.5.ConclusionOur FNSG mouse model provide an appropriate animal recipient for xenotransplantation.We can build both human immune system and liver system on it.The dual-rebuild mouse will give us more chance in immunology,regenerative medicine and oncology research.