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A construction method and application of an inducible b-cell deficient mouse model

A technology of mouse model and construction method, applied in the biological field, can solve problems such as unfavorable immune mechanism research, abnormal immune function, lack of mature B cells, etc.

Active Publication Date: 2022-01-18
广东南模生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of antibody heavy chain gene-deficient mouse model lacks mature B cells from birth. Based on the important role of mature B cells, its absence also leads to abnormal immune functions in mice, which is not conducive to the study of normal immune mechanisms.

Method used

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  • A construction method and application of an inducible b-cell deficient mouse model
  • A construction method and application of an inducible b-cell deficient mouse model
  • A construction method and application of an inducible b-cell deficient mouse model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cd19-DTR mouse model construction

[0031] In order to avoid destroying the expression of endogenous Cd19 in mice and causing B cell function defects under normal conditions, the inventor's Cd19-DTR mouse model was constructed and designed by inserting the IRES-DTR-EGFP element into the stop codon of the mouse Cd19 gene rear. The gene structure of wild-type Cd19 and the gene structure of Cd19-DTR gene modification are as follows: figure 1 shown.

[0032] 1) gRNA target site screening

[0033] The gRNA target sequence determines its targeting specificity and the efficiency of inducing Cas9 to cleave the target gene. The higher the efficiency of Cas9 cutting the target gene, the higher the efficiency of homologous recombination. Therefore, efficient and specific target sequence selection and design is the prerequisite for the successful construction of Cd19-DTR mouse model.

[0034] According to the recombination protocol, gRNAs (gRNA1-gRNA10) recognizing target site...

Embodiment 2

[0047] Detection of EGFP fluorescence expression in B cells of Cd19-DTR gene knock-in mice

[0048] During the construction of Cd19-DTR mice, DTR and EGFP were co-expressed in the form of fusion protein according to the design strategy. Therefore, when mouse B cells express DTR, the expression of EGFP fluorescence should be detected at the same time. The inventors detected the fluorescence expression of EGFP in peripheral blood B cells by flow cytometry. The results of flow cytometry were as Figure 5 As shown, almost all Cd19-positive cells in the peripheral blood of Cd19-DTR mice can detect EGFP fluorescence, and the fluorescence ratio is: 98.25% {1.16 / (1.16+65.24)}; Cd19-positive cells in the peripheral blood of wild-type C57BL / 6 mice EGFP fluorescence was almost undetectable in the cells.

Embodiment 3

[0050] Detection of B cell defect induced by diphtheria toxin in Cd19-DTR gene knock-in mice

[0051] In order to verify whether the expression of DTR in B cells can be eliminated by diphtheria toxin, the inventors administered diphtheria toxin to wild-type mice and Cd19-DTR mice by intraperitoneal injection on the 1st, 3rd, and 5th days of the experiment, respectively, at a dose of 0.015 mg / kg ; While administering diphtheria toxin, the B cell content in peripheral blood was detected on the 1st, 3rd, 7th, 14th, and 21st days of the experiment, and the relevant time points were as follows: Figure 6 Shown in A. The results of flow cytometry were as Figure 6 Shown in B: Cd19-DTR mice were intraperitoneally administered diphtheria toxin once, and on the third day Cd19 + The content of the cells dropped from 40-50% of normal to 1.26%; after the 3 administrations, the Cd19 + The proportion of cells is only 0.39%, and by the fourteenth day of the experiment, Cd19 + The cell ra...

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Abstract

The invention provides a method for constructing a B cell deficient mouse model and its application in B cell deficient drugs. This method inserts the diphtheria toxin receptor and green fluorescent protein into the Cd19 molecular gene expression cassette, and constructs a Cd19-IRES-DTR-EGFP (abbreviation: Cd19-DTR) gene-modified mouse model, which can realize intracellular The source Cd19 gene drives the expression of diphtheria toxin receptor and green fluorescent protein. Using this model, B cells can be killed by administering diphtheria toxin to mice at a specific time point or period of time, resulting in B cell defects, and the role of B cells in specific physiological and pathological processes can be studied.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of an inducible B cell deficient mouse model. Background technique [0002] B cells, also known as B lymphocytes, come from a kind of pluripotent stem cells derived from bone marrow. B cells can differentiate into plasma cells under antigen stimulation, and plasma cells can synthesize and secrete antibodies (immunoglobulins) to generate antibody-mediated humoral immune responses; at the same time, activated B cells are also a type of antigen-presenting cells whose surface BCR Combined with soluble antigen, after endocytosis and processing, it is presented to CD4 in the form of antigenic peptide: MHC-class II molecule complex + T cells. In addition, activated B cells can also produce a large number of cytokines, involved in immune regulation, inflammatory response and hematopoiesis. In recent years, in the study of tumor immunothera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90A01K67/027C12N15/62
CPCA01K67/0275C12N15/907C07K14/705A01K2217/05A01K2227/105A01K2267/03C07K2319/60
Inventor 孙瑞林王津津池骏
Owner 广东南模生物科技有限公司
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