基因修饰SLE,动物造模 z-bo 2022-09-13 408

　　Transgenic mouse model B6.Cg-Tg (Actb-TNFRSF6B) 754Jwu/J, gene knock-in mouse model 129S6.129X1 (B6)-Ptprctm1Weis / and many other types of transgenic mouse models for studying autoimmune diseases Have. J and B6.129X1-Ptprctm1Weis/J, gene knockout mouse model B6.129S6(Cg)-Cdkn1atm1Led/J, B6; 129S-Fcgr2btm1Ttk/J, conditional gene targeting mouse model B6.Cg-Rxratm1Krc/J and More than 20 strains. SLE is the inheritance of multiple genes. The current genetic modification technology only adds or knocks out one gene, so its phenotype is only partially simulated, which leads to the clinical manifestations and pathological changes of human SLE. very similar. In addition, the expression of gene products in different organs of the human body can produce different biological effects. In addition to the expected specific disease manifestations, the addition or deletion of genes may also cause other pathological changes. It partially clarified and explained the cause of SLE from the perspective of genetics. limit.

　　(I) Transgenic SLE mouse B6.Cg-Tg (Actb-TNF-RSF6B) 754Jwu/J

　　[Modeling mechanism] This mouse strain expresses the human tumor necrosis factor receptor superfamily 6B (TNFRSF6B) gene under the control of the human β-actin promoter. Female mice exhibit obvious lupus-like symptoms, which are characterized by lymphadenopathy, splenomegaly, anti-nuclear and anti-dsDNA antibodies, and glomerulonephritis. [Modeling method] The foreign gene components include human β-actin promoter, human TNFRSF6B cDNA and human β-actin poly A signal. Microinjection was used to inject foreign gene components into the male pronucleus of (C3HXC57BL/6) F1 mice. Then, the transgenic mice were backcrossed with wild-type C57BL/6 mice, and the foreign genes were introduced into the background of C57BL/6 mice. [[Characteristics of this model] The transgenic mouse developed an autoimmune disease similar to human SLE. Lymphadenopathy with abnormal cell phenotype and splenomegaly occurs between 4 and 6 months. This phenotype mainly occurs in women (64.5%), but only in 20% of men. Other symptoms include antinuclear and anti-dsDNA antibodies, increased abdominal B-1a subtypes, glomerulonephritis with IgG and C3 deposits, proteinuria, hematuria, and leukopenia. As the months increase, skin lesions, liver lymphocyte infiltration, anemia, white blood cell and thrombocytopenia appear. 63% of women and 81.8% of men survived for 14 months or longer.

　　[Evaluation and application of the model] This model can be used for the study of the etiology, etiology, immunomodulation of autoimmune diseases and the pharmacodynamics of SLE treatment drugs. In the experiment, C57BL/6 was used as a negative control.

　　(2) Knockout SLE mouse B6; 129S-Fcgr2btm1Ttk/J [Modeling mechanism] Low-affinity immunoglobulin receptor (FcγRII) is widely distributed in lymphocytes and bone marrow cells, but its biological learning effect is unknown. If B cells lack this receptor or its signal transduction pathway, it will lead to weakened feedback inhibition of immune complex-mediated antibody production. [Modeling method] Replace the "S2" exon of the 2.3 kb Fcgr2b (Fcreptor, IgG, lowaffinityⅡb) gene fragment of the first pair of mouse chromosomes with a targeting vector with a new gene cassette, and inactivate it. I will. This component is electroporated into ES cells. The genetic background of homozygous mutant mice is C57BL/6 and 129 hybrids. [Model Features] Homozygous mice of this strain develop normally, bone marrow and lymph nodes develop normally, cause antibody responses to thymus-dependent and thymus-independent antigens, and increase immunoglobulin. I will. Mice are sensitive to degranulation and passive skin allergic reactions caused by IgG. 40% of mice had proteinuria at 5 months of age, and 90% of mice had proteinuria at 9 months of age. The threshold of electrical shock stimulation is reduced, leading to generalized seizures. After an eight-month-old pathological examination, severe inflammation and multi-organ infiltration (lung, kidney, salivary gland, pancreas, liver, blood vessels, tongue, muscle), spleen and lymph nodes were enlarged, serum antinuclear antibody titer increased, and death showed At 9 months old. Arched back, edema, dehydration. [Model evaluation and application] Several phenotypes of this strain, such as autoantibodies, are similar to SLE and can be used to study the etiology and etiology of SLE. In addition, mice are susceptible to degranulation caused by IgG and prone skin allergic reactions (which can be used to study allergic reactions). B6129SF2/J was used as a negative control.

　　(3) Conditional knockout mouse B6.Cg-Rxratm1Krc/J

　　[Modeling mechanism] Retinoid X receptor (Rxr) is a member of the nuclear hormone receptor superfamily (retinoid). X-like receptor alpha (Rxm) is a nuclear receptor that is widely involved in the regulation of gene transcription activity. It forms homodimers alone or interacts with other nuclear receptors (retinoic acid receptor, thyroxine receptor, vitamin D receptor). , Peroxisome proliferator activated receptor and nerve growth factor induced receptor) form heterodimers and participate in cell signal transduction. Knockout of the xra gene will affect the transcriptional activity of many genes in the body. Knockout of the Rxra gene in bone marrow cell lines can cause autoimmune diseases.

　　[Modeling Method] Insert loxP sites upstream and downstream of the fourth exon of the Rxra target gene, and connect the downstream loxP sites to PGK-neor. This component is electroporated into ES cells and briefly transfected with Cre recombinase plasmid to remove the selection cassette. ES cells with loxP inserted on both sides of the 4th exon were injected into C57BL/6 blastocysts. The offspring are backcrossed with transgenic mice lacking a specific genetic background, which contains at least some C57BL/6 genetic elements. Use C57BL/6 to backcross at least 10 generations. Homozygous mice develop and reproduce normally, without any physiological or behavioral abnormalities. If you need a corresponding model, you need to mate this mouse with a specific Cre recombinase mouse, and the offspring will produce Rxra gene knockout in specific organs. After mating with Cre recombinase mice expressed in the liver, the offspring liver Rxra gene is knocked out, which can be used to study liver cell proliferation and liver regeneration. When mated with Cre recombinase mice expressed in bone marrow cell lines, the Rxra gene in the offspring bone marrow cell lines is knocked out, which can be used for glomerulonephritis, autoimmunity and SLE research.

　　[Characteristics of the model] Female mice aged 4-6 months have severe kidney disease. The kidney expands, the number of glomerular cells increases, and the number of apoptotic cells increases; the mesangial matrix of the glomerulus expands and thickens, IgG and IgM are deposited in large quantities, the mesangial cells proliferate, and the macrophages grow. Infiltration; ephron podocyte foot mutation and fusion. Proteinuria, the urine contains albumin. Antinuclear antibodies, anti-ssDNA, dsDNA and other autoantibodies in serum increase, while circulating creatine increases. Abdominal macrophages have defects in phagocytosis, low phagosome content and small prosthetic feet, which can impair the uptake of apoptotic cells and the aggregation of apoptotic cells.

　　[Evaluation and application of the model] This model is used to study glomerulonephritis, autoimmune diseases and SLE. In the experiment, C57BL/6 was used as a negative control. (4) Knock-in mouse CD45E613R [Modeling mechanism] CD45 is a receptor-like transmembrane protein tyrosine phosphatase expressed in all nucleated hematopoietic cells. The CD45E613R mutation causes the activation of polyclonal lymphocytes, which leads to increased autoantibodies, lymphatic proliferation, severe autoimmune nephritis and death. Both heterozygosity and homozygosity can develop this disease, indicating that CD45E613R is a dominant inheritance. [Modeling method] The glutamate 613 of the mouse CD45 gene was mutated to arginine (E613R) through gene targeted gene knock-in strategy. The mouse CD45 genome fragment glutamic acid 613 was mutated to arginine, and the neo box containing loxP on both sides was inserted into the downstream intron. This component is used to transfect ES cells from 129 sources, replacing endogenous CD45 sites with gene targeting. The homologous recombination rate is 3/1200 clones. Two of the clones were microinjected into C57BL/6 blastocysts for germline transmission. The offspring were crossed with β-actin Cre transgenic mice to remove the nail box. After mating with C57BL/6, the exogenous β-actin Cre gene was removed, and the mouse only carried the E613R mutation, with approximately 140 nucleotides inserted in the downstream intron. These mice are called CD45E613R mice. [Characteristics of the model] At the age of 3 months, the number of plasma cells in the spleen increased fourfold, and T1 cells and T28 cells increased. The γ-cells 6 months ago showed a slight increase in activation markers, and the activation markers of CD4 and CD8 T cells in vivo were slightly increased; CD8 T cells were highly sensitive to stimulation in vitro. Response. Double positive thymocytes are highly sensitive to stimulation in vitro. At 6 months, the CD4:CD8 T cell ratio ≥1.5, CD44hi, CD62Llo and memory T cells approximately doubled. Lymph nodes are enlarged and follicular B cells are highly sensitive to in vitro stimulation. At 12 months of age, inflammatory infiltrates were found around the liver and lung blood vessels of the mice.

　　[Model Evaluation and Application]

　　This mouse strain can be used for research on immunology and autoimmune diseases.

　　CD45E613R mice homozygous grow well. However, the phenotype depends on the genetic background. In the background of the 129X1/SvJ×C57BL/6 mutation, mice die as early as 15 weeks of age, while 43% of mice die before 50 weeks of age. .. The main pathological changes are abnormal activation of T cells and B cells, abnormal differentiation of granulocytes, hypertrophy of the spleen and lymph nodes, and glomerulonephritis. In the context of 129S6, B, T and bone marrow cells are sensitive to stimulation at the biomedical and cellular levels. B cell development is biased towards B1 cell line. A mild lymphoproliferative disease occurred.