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To construct T cell receptor β chain(TCRβ) gene knockout mouse model using CRISPR/Cas9 technology for genotype analysis and preliminary phenotypic characterization.
Tcrb-/- F0 generation mice were obtained by using CRISPR/Cas9 gene targeted knockout technology followed by embryo transfer. F1 generation mice were subsequently produced by mating sexually mature F0 Tcrb-/- mice with wild-type littermates. Genotypes were determined by polymerase chain reaction analysis of mouse tails. These results at the protein and cellular levels were validated using Western blot and flow cytometry, respectively.
The Tcrb gene knockout mice were successfully constructed and three genetically stable variants were obtained: wild-type(Tcrb+/+), heterozygous(Tcrb+/-), and homozygous knockout (Tcrb-/-) founder lines. Western blot analysis showed that TCRβ protein expression was barely detectable in the tissues of Tcrb-/- mice. Flow cytometry results indicated that compared with Tcrb+/+ mice, the numbers of CD4+ and CD8+ T cells in the peripheral blood, thymus and spleen of Tcrb+/- mice were significantly reduced, while in Tcrb-/- mice, CD4+ and CD8+ T cells were almost not expressed and the CD4+/CD8+ ratio was significantly reduced.
The Tcrb-/- mice were successfully constructed, which provides an experimental animal model for in-depth exploration of the pathological mechanisms of autoimmune diseases and the identification of potential drug targets.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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