Preliminary Study on the Function of miR9-2 in Immune Cell Development

This study used CRISPR/Cas9 technique to construct miR9-2 (microRNA9-2) knockout mice, and the effects of miR9-2 on B and T cells were identified by flow cytometry. By designing the sequences of sgRNA1 and sgRNA2 flanking mature miR9-2, and these two sgRNAs was ligated into linearized pX459 vectors respectively. Single-stranded sgRNA1, sgRNA2 in vitro transcription and Cas9 proteins were injected into male prokaryotes of fertilized eggs to obtain miR9-2 gene knockout mice. The changes of percentages and cell numbers of related immune cells in WT (Wild-type) mice and heterozygous (miR9-2+/−) mice were observed by flow cytometry. In this research, miR9-2+/− heterozygous mice model was successfully constructed. Flow cytometry analysis showed that the cells number of total B cells, CD4+ and CD8+ T cells in Sp (spleen) of heterozygous mice were all significantly decreased compared with WT mice. Compared with WT mice, the percentage and cell number of total B cells in BM (bone marrow) of heterozygous mice were both significantly decreased; the percentages and cells number of immature B cells, pro-pre B cells and pre B cells all decreased significantly, while the percentage of mature B cells significantly increased, and its cell number were comparable. In BM, the percentages of CD4+ and CD8+ T cells increased significantly, while their cells number were comparable in miR9-2+/− mice. In Th (thymus), the percentage of DN (double negative) cells increased significantly, and its cell number decreased significantly; the percentage and absolute number of DP (double positive) cells both decreased significantly; the percentages of CD4+ and CD8+ T cells increased significantly, while their cells number both decreased significantly. The research indicated that the knockout of miR9-2 affected B cells development in BM, T cells development in Th, and maturation and development of B and T cells in Sp to some extent.

CSTR: 32200.14.cjcb.2022.11.0006