PIKfyve基因敲除对小鼠成熟免疫细胞分布与数量的影响

Effect of PIKfyve gene deletion on distribution and number of mature immune cells in mice

目的建立PIKfyve基因全身与造血系统诱导性敲除小鼠模型,探索PIKfyve对免疫稳态的影响,为后续研究PIKfyve在造血系统中的功能提供基础。方法将LoxP位点插入PIKfyve基因第六外显子两端,建立PIKfyve+/flox小鼠,PIKfyve+/flox小鼠与PIKfyve+/flox小鼠互配得到PIKfyveflox/flox小鼠,PIKfyveflox/flox小鼠与UBC-Cre/ERT2小鼠杂交得到PIKfyve+/flox、UBC-Cre/ERT2小鼠,再与PIKfyveflox/flox小鼠杂交即可得到PIKfyveflox/flox、UBC-Cre/ERT2小鼠,即为目的小鼠。经基因型鉴定与诱导后,利用实时荧光定量PCR与Western印迹鉴定敲除效率,观察比较敲除小鼠与对照小鼠血象以及外周血、骨髓、脾脏中成熟免疫细胞的比例与数量。经同样配繁策略引入PIKfyveflox/flox、MX1-Cre小鼠对结果进行验证。结果通过小鼠杂交与鼠尾基因PCR鉴定成功获得PIKfyveflox/flox、UBC-Cre/ERT2小鼠,经诱导后敲除小鼠PIKfyve的mRNA与蛋白含量显著下降,表明模型小鼠建立成功。此外,外周血象检测发现PIKfyve基因缺失小鼠外周血中白细胞、淋巴细胞、中性粒细胞与单核细胞数量均显著下降。流式检测发现整体上看PIKfyve基因缺失小鼠B细胞与NK细胞比例显著下降,T细胞与中性粒细胞比例显著上升,而整体数量上依然呈现下降趋势。PIKfyveflox/flox、MX1-Cre小鼠实验结果与PIKfyveflox/flox、UBC-Cre/ERT2小鼠一致。结论成功建立PIKfyve敲除小鼠模型,发现PIKfyve调控成熟免疫细胞数量与比例,影响免疫稳态。

Objective To establish a mouse model with an inducible deletion of PIKfyve genes throughout the body and hematopoietic system and explore the impact of PIKfyve on immune homeostasis.Methods LoxP sites were inserted at both ends of exon 6 of the PIKfyve gene to establish PIKfyve+/flox mice.PIKfyve+/flox mice were cross-bred to produce PIKfyveflox/flox mice.PIKfyveflox/flox mice were cross-bred with UBC-Cre/ERT2 mice to obtain PIKfyve+/flox,UBC-Cre/ERT2 mice and PIKfyveflox/flox,UBC-Cre/ERT2 mice,the target mice.Genotyping was performed using tail gene PCR,and deletion efficiency was identified post-induction via real-time quantitative PCR(qPCR)and Western blotting.The proportion and number of mature immune cells in peripheral blood,bone marrow,and spleens were observed and compared between the knockout mice and control mice.A similar breeding strategy was adopted to introduce PIKfyveflox/flox,MX1-Cre mice to confirm the aforementioned results in a distinct mouse model.Results Successful generation of PIKfyveflox/flox,UBC-Cre/ERT2 mice was confirmed based on mouse breeding and tail gene PCR.After induction,a significant decrease in PIKfyve mRNA and protein contents in the knockout mice indicated the establishment of a proper model.Peripheral blood analysis revealed a significant reduction in white blood cells,lymphocytes,neutrophils,and monocytes in the PIKfyve-deficient mice.Flow cytometry demonstrated an overall decrease in the proportions of B cells and NK cells,with a significant increase in T cells and neutrophils,while the overall number trended down.Results from PIKfyveflox/flox,MX1-Cre mice were consistent with those of PIKfyveflox/flox,UBC-Cre/ERT2 mice.Conclusion A workable PIKfyve knockout mouse model has been established,suggesting that PIKfyve can regulate the number and proportions of mature immune cells while making a difference to immune homeostasis.