Construction And Phenotypic Analysis Of The Nrf2/CCK2R Double-gene Knockout Mouse Model

Studies showed that Nuclear factor(Nuclear factor(erythroid-derived 2)-like2,NF-E2-Related Factor2,Nrf2,NFE2L2)is an important transcription factor in organisms,which can control the expression of a variety of antioxidant enzymes and protective enzymes in human body.Cholecys-tokinin Receptor 2(also known as CCK2R,CCK2R,CCKBR)regulates the gastrointestinal tract,is involved in pain relief,panic regulation,learning and memory feeding behavior regulation,and mediates growth effect.Existed studies have shown that neuropathic diseases or inflammatory pathological states can affect the endocrine regulation axis,lead to immune function abnormalities,and easily induce autoimmune diseases.Experimental Purpose:In this paper,CRISPR/Cas9 gene editing technology was used to complete Nrf2/CCK2R double gene knockout based on C57BL/6J mice,and it was identified and propagated in SPF environment,so as to establish a scientific and rigorous Nrf2/CCK2R double gene knockout mouse model,which laid a solid foundation for the later establishment of inflammatory immune diseases.The experimental methods were as follows:(1)Nrf2/CCK2R double gene knockout mice were constructed with C57BL/6J mice using CRISPR/Cas9 gene editing technology,and homozygous mice were obtained by breeding and identification.(2)Homozygous mice were propagated in SPF environment to obtain a certain number of Nrf2/CCK2R double knockout mouse inbred lines;(3)Phenotypic analysis of Nrf2/CCK2R double-gene knockout mice,which had been bred for 3-5 generations,was performed on the inbred mice,such as organ coefficient,routine analysis of blood,blood biochemical examination,HE staining pathological observation,immunoflow characteristics and inflammatory immune factor ELISA assay.Results:(1)Nrf2/CCK2R double gene knockout mouse model was successfully constructed;(2)Four generations of 157 homozygous mouse progeny were successfully bred,and Nrf2/CCK2R double gene knockout had a certain effect on the growth and breeding of mice;(3)Organ coefficient results showed that the spleen,liver,stomach and kidney of 6-8 weeks old double-tapped mice were significantly lower than those of wild-type mice of the same week,while the stomach of 4-6 months old double-tapped mice was significantly lower than that of wild-type mice of the same week.The results of blood routine showed that many blood routine indexes were abnormal in 6-8 weeks old double-tap mice,and only MCV(mean red blood cell volume)was abnormal in 4-6months old mice.Blood biochemical results showed that AST and BUN of liver function were significantly different from those of wild-type mice.HE pathology showed that there was no difference in the level of organs in the histopathologic sections of each group.Immunoflow analysis showed that the proportions of CD3~+CD4~+(THL cell),CD3~+CD8~+(CTL cell),CD4~+CD8~+(DPT cell)and CD3~-CD19~+(B cell)in spleen of 4 to 6 months old mice were significantly different from those of wild-type mice of the same month.Inflammatory immune factor ELISA assay showed that serum levels of IL-1β,IL-6,IL-10,NF-κB,TNF-αand JNK1 in DKO groups were increased to varying degrees.Conclusion:Nrf2/CCK2R double gene knockout has different effects on the growth and development of mice,immune characteristics,inflammatory immune factors,These results suggested that the mice had immune deficiency and disease susceptibility,may provides inflammatory internal environment for the occurrence and development of inflammatory immune diseases,which can be used for further experimental modeling in inflammatory immune diseases.