| Immunodeficient animals play an important role in the study of the pathogenesis and treatment of human immunodeficiency diseases,as well as for the construction of animal models of tumors and humanized animal models.Recombination-activating genes(RAG)play an important role in V(D)J recombination.The knockout of RAG2 gene knock out(KO)can prevent lymphocyte differentiation,resulting in a lack of mature T and B cells.The development of immune system and the mechanism of immune response in pigs are more similar to that in humans,and they are useful tools for biomedical research.Therefore,it is of great significance to construct Rag2 KO immunodeficient pig model for its further research and application.In our previous studies,we used Diannan miniature pigs as the experimental animal to obtain the RAG2 KO fetus through CRISPR/Cas9 gene editing and somatic cell nuclear transfer.Then the RAG2 KO fibroblast cell line was isolated from RAG2 KO fetuses.In this study,RAG2 KO fibroblast cells were used as donor cells for the second round cloning to obtain RAG2 KO gene edited pigs.The genotype and effects of RAG2 KO on V(D)J rearrangement was verified by PCR and TA.Meanwhile,the effects of RAG2 knockdown on the development of immune organs and immune cells in pigs were investigated by gross observation and HE staining of thymus and spleen.The phenotype of peripheral blood immune cells were tested by flow cytometry and blood routine testing.The cloned embryos were surgically transferred to 4 surrogate sows with a total of 2680 embryos.Two of the 4 surrogate sows were pregnant,with a pregnancy rate of 50%.After 116 d of gestation,the 2 gestating sows gave birth to 12live piglets.DNA was extracted from muscle tissues of 12 piglets and their genotypes were identified by PCR,and all 12 piglets were found to be positive.The size of thymus and spleen of RAG2 KO pigs were significantly decreased than those of wild-type(WT)Diannan miniature pigs,and the HE results showed that the thymus tissue of WT pigs showed obvious germinal centers and thymic vesicles,while the thymus tissue of RAG2 KO pigs was disorganized,and the characteristic structure of thymic vesicles in the thymus was not obvious,and no obvious germinal centers were seen.The spleen tissue structure was more disorganized in RAG2 KO pigs compared with WT pig,and the central arterial canal in the splenic red medulla disappearing and the transition of the marginal zone blurred in the RAG2 KO piglets.The peripheral blood routine test results in RAG2 KO pig,showed that the total leukocyte and lymphocyte counts were significantly decreased(p<0.05)and eosinophils were significantly increased(p<0.05),while the numbers of basophils,neutrophils and monocytes were not significantly changed(p>0.05)compared with the wild type.The results suggest that RAG2 knockdown can down-regulate the number of lymphocytes.The results of flow cytometry showed that the percentage of mature B lymphocyte in RAG2 KO pigs was significantly lower than that in WT,and the percentage of mature T lymphocyte in RAG2 KO pigs was significantly lower than that in WT.In conclusion,RAG2 KO piglets were successfully obtained in this study,and the size of thymus and spleen were significantly decreased.In RAG2 KO pigs,there were no obvious thymic vesicles and germinal centers,and T cell numbers decreased in the thymus.And the pathological and histological examination of RAG2 KO pig showed that the central arterial canal in the splenic red marrow disappeared and the withblurred transition in the marginal zone.And in peripheral blood of RAG2 KO pigs,the proportion of mature T and B lymphocytes significantly decreased than that of WT pig.These successfully indicated that RAG2 KO led to the development defects of immune organs and immune cells.The above results successfully verified the immunodeficiency phenotype of RAG2 KO pigs,and this study may provide experimental animals for the study of immunodeficiency diseases and the construction of tumor models and humanized animals. |
PDF Full Text Request