Establishment of mouse model of cortical expansion and sulcus formation
A mouse, cerebral cortex technology, applied in the field of neurobiology
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Embodiment 1
[0058] Example 1. Sorting of different types of neural stem cells in the cerebral cortex
[0059] 1) Obtain brain tissue, separate the left and right hemispheres, and cut out the position of the frontal lobe according to the brain atlas for shock slices. Afterwards, the ventricular area, extraventricular area and cortical area were finely segmented under the microscope.
[0060]2) Digest tissues in different regions with papain at 37° C. for 20 minutes, and gently blow with a pipette to form a single cell suspension. The reaction was neutralized with DMEM complete medium containing 5% fetal bovine serum (FBS), centrifuged at 2000 rpm for 5 minutes, and resuspended with PBS containing 0.5% serum albumin (BSA). The single cell suspension in different regions was added with flow cytometric antibodies in proportion: CD15 (eBioscience), CD184 (BD Biosciences), CD56 (BD Biosciences), CD140α (BD Biosciences). CD15-positive / CD184-positive / CD56-negative / CD140α-negative cells were col...
Embodiment 2
[0061] Example 2. TMEM14B is a gene specifically expressed in human brain oRG cells
[0062] 1) The quality of the results of single-cell sequencing was tested by means of clustering [4].
[0063] 2) Remove the genes with very low expression in all cell types, and then according to the significant difference in gene expression in different cells, we screen out 9 genes specifically expressed in oRG, among which TMEM14B, as a gene specifically expressed in oRG cells, only exists in In primates, and the verification of in situ hybridization also proved that the gene specifically exists in the extraventricular region ( figure 2 ).
Embodiment 3
[0064] Example 3. TMEM14B transgenic mice showed a significant increase in the number of neural stem cells.
[0065] 1) Preparation of transgenic mice. Human TMEM14B gene knock-in mice (Biocytogen) and NestinCre mice were used to obtain the first-generation NESCre::T / + offspring, and their embryonic stages were analyzed for phenotype[1-3]. Experiments found that the embryonic transgenic cortex was significantly thickened, and both IP and oRG neural stem cells were significantly increased ( image 3 A).
[0066] 2) Western Blot analysis of phenotype. The cortex of embryonic transgenic and normal mice was taken for total protein extraction, and then the expression of different proteins such as Sox2, Pax6, Tbr2 was detected, and it was found that the expression of neural stem cell-specific proteins in the cortex of transgenic mice was significantly increased ( image 3 B).
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